Humanized monoclonal antibodies that target VE-PTP (HPTP-β)

ABSTRACT

The disclosure provides compositions and methods for the treatment of ocular conditions associated with angiogenesis comprising administering an antibody that targets a tyrosine phosphatase inhibitor in a subject.

CROSS REFERENCE

This application is a continuation of U.S. application Ser. No.16/180,850, filed on Nov. 5, 2018, which is a continuation of U.S.application Ser. No. 15/654,289, filed on Jul. 19, 2017, now U.S. Pat.No. 10,253,094, issued on Apr. 9, 2019, which claims the benefit of U.S.Provisional Application No. 62/364,381, filed Jul. 20, 2016, and U.S.Provisional Application No. 62/377,072, filed Aug. 19, 2016, each ofwhich is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 28, 2020, isnamed 45725724303 SL.txt and is 131,588 bytes in size.

BACKGROUND OF THE INVENTION

Monoclonal antibodies generated from non-human organisms can elicit animmune response when administered to humans. Segments of these non-humanmonoclonal antibodies can be replaced with humanized sequences through ahumanization process to reduce the likelihood of drug immunogenicitywhile preserving target specificity.

INCORPORATION BY REFERENCE

Each patent, publication, and non-patent literature cited in theapplication is hereby incorporated by reference in its entirety as ifeach was incorporated by reference individually.

SUMMARY OF THE INVENTION

In some embodiments, the invention provides a compound comprising asequence that is at least 80% identical to SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, or SEQ ID NO: 12.

In some embodiments, the invention provides a compound comprising asequence that is at least 80% identical to SEQ ID NO: 20, SEQ ID NO: 21,SEQ ID NO: 22, or SEQ ID NO: 23.

In some embodiments, the invention provides a compound comprising: a) aheavy chain that comprises a sequence that is at least 80% identical toSEQ ID NO: 30; and b) a light chain that comprises a sequence that is atleast 80% identical to SEQ ID NO: 34.

In some embodiments, the invention provides a compound comprising: a) aheavy chain that comprises a sequence that is at least 80% identical toSEQ ID NO: 29; and b) a light chain that comprises a sequence that is atleast 80% identical to SEQ ID NO: 35.

In some embodiments, the invention provides a compound comprising: a) aheavy chain that comprises a sequence that is at least 80% identical toSEQ ID NO: 30; and b) a light chain that comprises a sequence that is atleast 80% identical to SEQ ID NO: 37.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a sequence alignment of the murine V_(H) sequence(truncated V_(H0); SEQ ID NO: 47) with the four humanized variants SEQID NO: 9 (V_(H1)), SEQ ID NO: 10 (V_(H2)), SEQ ID NO: 11 (V_(H3)), andSEQ ID NO: 12 (V_(H4)).

FIG. 2 shows a sequence alignment of the murine V_(L), sequence(truncated V_(L0); SEQ ID NO: 48) with the four humanized V_(L) variantsSEQ ID NO: 20 (V_(L1)), SEQ ID NO: 21 (V_(L2)), SEQ ID NO: 22 (V_(L3)),and SEQ ID NO: 23 (V_(L4)).

FIG. 3 summarizes the hybridoma technology used to generate monoclonalantibodies against the HPTP-β extracellular domain (human VE-PTP/HPTP-βECD). The 6-HIS tag disclosed in FIG. 3 is SEQ ID NO: 52.

FIG. 4 illustrates an immunoprecipitation western blot (left panel) andflow cytometry results (right panel) of R15E6 bound to endogenous humanVE-PTP (HPTP-β) in human endothelial cells.

FIG. 5 illustrates the binding specificity of R15E6 to recombinant,6-His tagged human VE-PTP (HPTP-β) (The “6-His” disclosed in FIG. 5 isSEQ ID NO: 52), cynomolgus PTP-β, and human PTP-η extracellular domainproteins by western blot.

FIG. 6 illustrates a western blot (panel A) and quantification (panel B)of concentration-dependent phosphorylation of Tie2 and enhancedendothelial cell viability (panel C) by R15E6.

FIG. 7 illustrates effects of rat anti-mouse VE-PTP monoclonal antibody109.1 on Tie2 (top left panel), mouse VE-PTP expression (bottom leftpanel), and Akt (right panel) in mouse endothelial cells as determinedby western blot.

FIG. 8 illustrates Tie2 activation by polyclonal antibodies againstmouse VE-PTP ECD after 1 hour of incubation (panel A) and 3 min ofincubation (panel B) in mouse endothelial cells.

FIG. 9 illustrates in vitro inhibition of thrombin- and VEGF-inducedendothelial cell permeability by mouse VE-PTP ECD polyclonal antibodies.

FIG. 10 illustrates in vivo inhibition of VEGF-induced cutaneousvascular permeability by intravenous administration of monoclonalantibody 109.1 (panel A) and mouse VE-PTP ECD polyclonal antibodies(PTP1-8) (panel B), and immunoprecipitation of Tie2 from lung lysates ofcontrol IgG (−) or anti-VE-PTP antibody-injected mice (+) (panel C).

FIG. 11 depicts effects on retinal neovascularization (panels A and B)and choroidal neovascularization (panel C) after intraocularadministration of anti-mouse VE-PTP monoclonal antibody 109.1.

FIG. 12 shows quantification of the mean area of retinalneovascularization (panels A and B) and choroidal neovascularization(panel C) after intraocular administration of an anti-mouse VE-PTPmonoclonal antibody 109.1.

FIG. 13 depicts a reducing (panel A) and a non-reducing (panel B)SDS-PAGE of anti-human VE-PTP (anti-HPTP-β) humanized variants of R15E6.

FIG. 14 illustrates binding of HC1 variants of R15E6 and HC0LC0 toHPTP-β.

FIG. 15 illustrates binding of HC2 variants of R15E6 and HC0LC0 toHPTP-β.

FIG. 16 illustrates binding of HC3 variants of R15E6 and HC0LC0 toHPTP-β.

FIG. 17 illustrates binding of HC4 variants of R15E6 and HC0LC0 toHPTP-β.

FIG. 18 illustrates Tie2 phosphorylation (left panel) and Akt activation(right panel) in the absence of Ang1 by the various humanized antibodiesas determined by western blot.

FIG. 19 illustrates concentration-dependent Tie2 activation (top panel)and Akt activation (bottom panel) in the absence of ligands by HC2LC4and HC2LC1 as determined by western blot.

FIG. 20 illustrates Tie2 phosphorylation (left panel) and Akt activation(right panel) in the presence of Ang1 and Ang2 by HC2LC4 and HC2LC1 asdetermined by western blot.

FIG. 21 illustrates Tie2 phosphorylation (panel A), human VE-PTP(HPTP-β) expression (panel B), and Akt activation (panel C) in thepresence of Ang1 and/or Ang2 by HC2LC1 and HC2LC4 as determined bywestern blot.

FIG. 22 illustrates Akt activation by HC2LC1 alone and with excessrecombinant human VE-PTP extracellular domain protein (βECD 6His (The“6-His” disclosed in FIG. 22 is SEQ ID NO: 52) or βECD Fc) as determinedby western blot.

FIG. 23 illustrates enhanced endothelial cell viability by HC2LC1 andHC0LC0 as compared to the parenteral antibody (R15E6) and a mouse IgG1control.

FIG. 24 illustrates enhanced efficacy of an anti-mouse VE-PTPantibody/aflibercept combination therapy for the treatment of retinaldetachment in a mouse model.

FIG. 25 illustrates that subcutaneous administration of monoclonalantibody 109.1 reduced ischemic neovascularization in mice compared tovehicle.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides compositions and methods for targetingvascular endothelial protein tyrosine phosphatase (VE-PTP or VEPTP) orhuman protein tyrosine phosphatase-beta (HPTP-β) for the treatment ofocular disorders that are characterized by, for example, vascularinstability, angiogenesis, neovascularization, vascular leakage, andedema. Compositions disclosed herein can activate Tie2 signaling bypromoting protein phosphorylation, such as phosphorylation of the Tie2protein.

VE-PTP is a member of the receptor-like family of the protein tyrosinephosphatases (PTPases). VE-PTP is a transmembrane protein foundprimarily in vascular endothelial cells that displays structural andfunctional similarity to cell adhesion molecules. VE-PTP is found invarious species including, for example, zebrafish, chicken, dog, mouse,marmoset, monkey, and human. The human orthologue of VE-PTP is HPTP-β.

Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factorhomology domains 2) is a membrane receptor tyrosine kinase expressedprimarily in vascular endothelial cells throughout development. Theprinciple regulators of Tie2 phosphorylation are angiopoietin 1 (Ang1)and angiopoietin 2 (Ang2). Ang1 is an agonist of Tie2, and binding ofAng1 to Tie2 promotes receptor phosphorylation. Ang2 acts in acontext-dependent antagonist or agonist of Tie2. Binding of Ang1 to Tie2increases the level of endogenous Tie2 receptor phosphorylation andinitiates downstream signaling to induce vascular stabilization throughhighly organized angiogenesis, tightening of the endothelial celljunctions (endothelial cell proximity), enhanced endothelial viability,reduced endothelial inflammation and improved endothelial function.During angiogenesis, Ang2 acts as a negative regulator of Ang1-Tie2signaling.

Under physiological conditions, the duration of Tie2 phosphorylation isregulated by VE-PTP (HPTP-β), which removes the phosphate from the Tie2receptor. By inhibiting VE-PTP (HPTP-β), the level of Tie2phosphorylation substantially increases, restoring vascular stability. AVE-PTP (HPTP-β) inhibitor, for example, an antibody that binds VE-PTP(HPTP-β), can activate Tie2 downstream signaling by inhibiting VE-PTP(HPTP-β). Inhibition of VE-PTP (HPTP-β) by the inhibitor can providevascular stability in subjects with ocular disorders described herein.

A VE-PTP (HPTP-β) inhibitor of the disclosure can comprise the murinemonoclonal antibody R15E6 produced by hybridoma cell line ATCC No.PTA-7580, which is immunoreactive to the extracellular domain of humanVE-PTP (HPTP-β) (SEQ ID NO. 45), is immunoreactive to the first FN3repeat of human VE-PTP (HPTP-β) (SEQ ID NO. 46). The VE-PTP (HPTP-β)inhibitor can comprise an antibody having the same or substantially thesame biological characteristics of R15E6, an antibody fragment of R15E6,wherein the fragment comprises one or both of the heavy and light chainvariable regions, a F(ab′)2 of R15E6, dimers or trimers of a Fab, Fv,scFv, and dia-, tria-, or tetrabodies derived from R15E6. In someembodiments, a fragment of a compound described herein can be assayedfor any biological activity described herein. In some embodiments, afragment of a compound described herein can activate Tie2, or providethe biological activity of the corresponding intact antibody at anequipotent, increased, or decreased level.

Extracellular domain of human VE-PTP (HPTP-β) (SEQ ID NO: 45):MLSHGAGLALWITLSLLQTGLAEPERCNFTLAESKASSHSVSIQWRILGSPCNFSLIYSSDTLGAALCPTFRIDNTTYGCNLQDLQAGTIYNFRIISLDEERTVVLQTDPLPPARFGVSKEKTTSTSLHVWWTPSSGKVTSYEVQLFDENNQKIQGVQIQESTSWNEYTFFNLTAGSKYNIAITAVSGGKRSFSVYTNGSTVPSPVKDIGISTKANSLLISWSHGSGNVERYRLMLMDKGILVHGGVVDKHATSYAFHGLTPGYLYNLTVMTEAAGLQNYRWKLVRTAPMEVSNLKVTNDGSLTSLKVKWQRPPGNVDSYNITLSHKGTIKESRVLAPWITETHFKELVPGRLYQVTVSCVSGELSAQKMAVGRTFPDKVANLEANNNGRMRSLVVSWSPPAGDWEQYRILLFNDSVVLLNITVGKEETQYVMDDTGLVPGRQYEVEVIVESGNLKNSERCQGRTVPLAVLQLRVKHANETSLSIMWQTPVAEWEKYIISLADRDLLLIHKSLSKDAKEFTFTDLVPGRKYMATVTSISGDLKNSSSVKGRTVPAQVTDLHVANQGMTSSLFTNWTQAQGDVEFYQVLLIHENVVIKNESISSETSRYSFHSLKSGSLYSVVVTTVSGGISSRQVVVEGRTVPSSVSGVTVNNSGRNDYLSVSWLLAPGDVDNYEVTLSHDGKVVQSLVIAKSVRECSFSSLTPGRLYTVTITTRSGKYENHSFSQERTVPDKVQGVSVSNSARSDYLRVSWVHATGDFDHYEVTIKNKNNFIQTKSIPKSENECVFVQLVPGRLYSVTVTTKSGQYEANEQGNGRTIPEPVKDLTLRNRSTEDLHVTWSGANGDVDQYEIQLLFNDMKVFPPFHLVNTATEYRFTSLTPGRQYKILVLTISGDVQQSAFIEGFTVPSAVKNIHISPNGATDSLTVNWTPGGGDVDSYTVSAFRHSQKVDSQTIPKHVFEHTFHRLEAGEQYQIMIASVSGSLKNQINVVGRTVPASVQGVIADNAYSSYSLIVSWQKAAGVAERYDILLLTENGILLRNTSEPATTKQHKFEDLTPGKKYKIQILTVSGGLFSKEAQTEGRTVPAAVTDLRITENSTRHLSFRWTASEGELSWYNIFLYNPDGNLQERAQVDPLVQSFSFQNLLQGRMYKMVIVTHSGELSNESFIFGRTVPASVSHLRGSNRNTTDSLWFNWSPASGDFDFYELILYNPNGTKKENWKDKDLTEWRFQGLVPGRKYVLWVVTHSGDLSNKVTAESRTAPSPPSLMSFADIANTSLAITWKGPPDWTDYNDFELQWLPRDALTVFNPYNNRKSEGRIVYGLRPGRSYQFNVKTVSGDSWKTYSKPIFGSVRTKPDKIQNLHCRPQNSTAIACSWIPPDSDFDGYSIECRKMDTQEVEFSRKLEKEKSLLNIMMLVPHKRYLVSIKVQSAGMTSEVVEDSTITMIDRPPPPPPHIRVNEKDVLISKSSINFTVNCSWFSDTNGAVKYFTVVVREADGSDELKPEQQHPLPSYLEYRHNASIRVYQTNYFASKCAENPNSNSKSFNIKLGAEMESLGGKCDPTQQKFCDGPLKPHTAYRISIRAFTQLFDEDLKEFTKPLY SDTFFSLPITTESEPLFGAIEFirst FN3 repeat of human VE-PTP (HPTP-β) (SEQ ID NO: 46):LAEPERCNFTLAESKASSHSVSIQWRILGSPCNFSLIYSSDTLGAALCPTFRIDNTTYGCNLQDLQAGTIYNFRIISLDEERTVVLQTD

A VE-PTP (HPTP-β) inhibitor of the disclosure can include an antibody,or an antibody fragment, variant, or derivative thereof, either alone orin combination with other amino acid sequences. The inhibitor canundergo modifications, for example, enzymatic cleavage, andposttranslational modifications.

A VE-PTP (HPTP-β) inhibitor of the disclosure can bind adominant-negative isoform of VE-PTP (HPTP-β). In some embodiments, thisdominant-negative isoform can correspond to a form of VE-PTP (HPTP-β)deficient in phosphatase activity that can compete with endogenousVE-PTP (HPTP-β). Functional assessment of dominant-negative VE-PTP(HPTP-β) can occur via delivery of the transgene and determination ofthe effect on Tie2 phosphorylation.

A VE-PTP (HPTP-β) inhibitor of the disclosure can comprise a pluralityof VE-PTP (HPTP-β) binding sites. In some embodiments, a VE-PTP (HPTP-β)inhibitor can bind to two VE-PTP (HPTP-β) molecules simultaneously,thereby bringing the two VE-PTP (HPTP-β) molecules into close proximity.A VE-PTP (HPTP-β) inhibitor can bind to three VE-PTP (HPTP-β) moleculessimultaneously, thereby bringing the three VE-PTP (HPTP-β) moleculesinto close proximity.

A VE-PTP (HPTP-β) inhibitor of the disclosure can be covalently ornon-covalently conjugated to another moiety or vehicle. A moiety orvehicle can, for example, inhibit degradation, increase half-life,increase absorption, reduce toxicity, reduce immunogenicity, and/orincrease biological activity of the inhibitor. Non-limiting examples ofthe moiety include Fc domains of immunoglobulins, polymers such aspolyethylene glycol (PEG), polylysine, and dextran, lipids, cholesterolgroups such as steroids, carbohydrates, dendrimers, oligosaccharides,and peptides.

The compounds of the present invention can be used for targeting VE-PTP(HPTP-β) to restore Tie2 activity and initiate downstream signalingcascades including, for example, Akt/PI3-K signaling, Rac1 signaling,MAPK/Ras signaling. In some embodiments, compounds of the presentinvention can inhibit NF-κB signaling. The activation of Tie2 can leadto vascular stabilization, which can be beneficial for the treatment ofdiabetes-related conditions and ocular conditions including, forexample, retinopathy, diabetic retinopathy, retinal perfusion, ocularneovascularization, ocular vascular leak, ocular edema, intraocularpressure, ocular hypertension, ocular inflammation, glaucoma, and ocularhemorrhage. The compounds can also be effective for the treatment ofperipheral artery disease including, for example, wound healing,destabilized blood flow, cardiac fibrosis, erectile dysfunction,cardiomyopathy, ischemic injury, cardiac hypertrophy, interstitialfibrosis, nephropathy, albuminuria, glomerulosclerosis, renal fibrosis,neuropathy, and neuronal inflammation.

Antibodies

Antibodies comprise of two identical heavy chain (H) polypeptidesequences and two identical light chain (L) polypeptide sequences. Eachof the heavy chains comprises one N-terminal variable (V_(H)) region andthree C-terminal constant (C_(H)1, C_(H)2, and C_(H)3) regions. Each ofthe light chains comprises one N-terminal variable (V_(L)) region andone C-terminal constant (C_(L)) region. The light chain variable regionis aligned with the heavy chain variable region and the light chainconstant region is aligned with heavy chain constant region C_(H1). Thepairing of a heavy chain variable region and light chain variable regiontogether forms a single antigen-binding site. Each light chain is linkedto a heavy chain by one covalent disulfide bond. The two heavy chainsare linked to each other by one or more disulfide bonds depending on theheavy chain isotype. Each heavy and light chain also comprisesregularly-spaced intrachain disulfide bridges.

The light chain from any vertebrate species can be designated kappa orlambda based on the amino acid sequences of the constant region.Depending on the amino acid sequence of the constant region of the heavychains, immunoglobulins can be categorized into five classes ofimmunoglobulins (IgA, IgD, IgE, IgG, and IgM), each having heavy chainsdesignated alpha, delta, epsilon, gamma, and mu, respectively. The alphaand gamma classes are further divided into subclasses on the basis ofdifferences in the sequence and function of the heavy chain constantregion. Subclasses of IgA and IgG expressed by humans include IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2.

A variable (V) region comprises segments that can differ extensively insequence among antibodies. The variable region mediates antigen-bindingand defines specificity of a particular antibody for its antigen.However, the variability is not evenly distributed across the span ofthe variable regions. Instead, the variable regions consist ofrelatively invariant stretches called framework regions (FR) of 15-30amino acids separated by shorter regions of extreme variability calledhypervariable regions that are each 9-12 amino acids long. The variableregions of native heavy and light chains each comprise four frameworkregions, largely adopting a f3-sheet configuration, connected by threehypervariable regions, which form loops connecting, and in some casesforming a part of, the f3-sheet structure. The hypervariable regions ineach chain are held together in close proximity by the framework regionsand, with the hypervariable regions from the other chain, contribute tothe formation of the antigen-binding site of antibodies. The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody dependent cellular cytotoxicity (ADCC).

A hypervariable region can comprise amino acid residues from acomplementarity determining region (CDR), for example, around aboutresidues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chainvariable region, and around about 1-35 (H1), 50-65 (H2), and 95-102 (H3)in the heavy chain variable region, and/or residues from a hypervariableloop.

A monoclonal antibody can be obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that can be present in minor amounts. In contrast topolyclonal antibody preparations, which include different antibodiesdirected against different epitopes, each monoclonal antibody isdirected against a single epitope. In addition to the specificity, themonoclonal antibodies are advantageous in that each can be synthesizeduncontaminated by other antibodies.

The monoclonal antibodies used herein can be, for example, chimericantibodies wherein a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as antigen-binding fragments of such antibodies.

An antibody fragment can comprise a portion of a multimeric antibody,for example, the antigen-binding or variable region of the intactantibody. Non-limiting examples of antibody fragments include Fab, Fab′,F(ab′)₂, dimers, and trimers of Fab conjugates, Fv, scFv, minibodies,dia-, tria-, and tetrabodies, and linear antibodies.

Non-limiting examples of epitopes include amino acids, sugars, lipids,phosphoryl, and sulfonyl groups. An epitope can have specificthree-dimensional structural characteristics, and/or specific chargecharacteristics. Epitopes can be conformational or linear.

Humanized Monoclonal Antibodies

Suitable antibodies that target VE-PTP can be identified using a varietyof techniques. For example, candidate agents can be screened for bindingto VE-PTP. Agents that bind to VE-PTP can be screened for activity, forexample, inhibition of VE-PTP-mediated dephosphorylation of Tie2. Insome embodiments, the candidate agents are first screened in vivo foractivity.

The selection of a suitable assay for use in identification of aspecific inhibitor depends on the nature of the candidate agent to bescreened. For example, where the candidates are antibodies orpeptibodies, which comprise an Fc moiety, fluorescence-activated cellsorting (FACS) analysis allows the candidate agent to be selected basedon the ability to bind to a cell that expresses VE-PTP. The cell canendogenously express VE-PTP or can be genetically engineered to expressVE-PTP. For other candidate agents such as aptamers, other techniquescan be utilized. For example, aptamers that specifically bind to VE-PTPcan be selected using systematic evolution of ligands by exponentialenrichment (SELEX), which selects specific aptamers through repeatedrounds of in vitro selection.

VE-PTP inhibitors can be screened for VE-PTP-mediated activity, forexample, inhibition of Tie2 dephosphorylation. In one suitable assaybased on western blotting, human umbilical vein endothelial cells(HUVEC) are cultured in serum free media in the presence or absence ofvarious concentrations of candidate agent, and lysates of the cells areprepared, immunoprecipitated with a Tie2 antibody, resolved bypolyacrylamide gel electrophoresis (SDS-PAGE), and transferred to apolyvinylidene difluoride (PVDF) membrane. Membrane-boundimmunoprecipitated proteins are then serially western blotted with ananti-phosphotyrosine antibody to quantify Tie2 phosphorylation followedby a Tie2 antibody to quantify total Tie2. Tie2 phosphorylation isexpressed as the ratio of the anti-phosphotyrosine signal over the totalTie2 signal. Greater levels of the anti-phosphotyrosine signal indicategreater VE-PTP inhibition by the candidate agent.

Hybridoma technology is a method for producing large numbers of amonoclonal antibody targeting a specific antigen. Hybridoma developmentbegins by injecting a mammalian host, such as a mouse (murine) orrabbit, with an antigen that provokes an immune response. In response tothe antigen, B-lymphocytes (B-cells) in the host produce antibodies thatbind to the antigen. The B-cells are then harvested from the host andfused with immortal B cell cancer cells (myeloma cells) to produce ahybrid cell line known as a hybridoma. The hybridoma retains both theantibody-producing ability of the B cell and the exaggerated longevityand reproductivity of the myeloma. The hybridomas can be grown inculture starting with one viable hybridoma cell to produce new culturesconsisting of genetically identical hybridomas that, in turn, produceone antibody per culture (monoclonal) or mixtures of differentantibodies (polyclonal). The myeloma cell line used in this process isselected for the ability to grow in tissue culture and for the inabilityto synthesize antibodies. After culturing, a primary screening processis performed to identify and select the hybridomas that produceantibodies with highest specificity. Non-limiting examples of antibodyscreening techniques include ELISA and immunocytochemical screening. Thelead hybridomas identified from screening can then be characterized forreactivity, binding affinity, specificity, and cross-reactivity.

In some embodiments, the binding affinity (K_(D)) to HPTP-β of acompound of the disclosure is from about 70 pM to about 70 nM, 1 nM toabout 70 nM, or at least as strong as about 1 nM. In some embodiments,the binding affinity (K_(D)) to HPTP-β of a compound of the disclosureis from about 4 nM to about 70 nM.

Other recombinant antibody engineering techniques involve the use ofviruses or yeast rather than mammals. These techniques rely on rapidcloning of immunoglobulin gene segments to create antibody librarieswith slightly different amino acid sequences from which antibodies withdesired specificities can be selected. Antibody libraries can be used toenhance specificity to target antigens, stability in variousenvironmental conditions, therapeutic efficacy, and detectability indiagnostic applications.

For human administration, monoclonal antibodies generated from non-humanspecies can be further optimized by a humanization process to reduce thelikelihood of immunogenicity while preserving target specificity.Humanization processes involve the incorporation of human DNA to thegenetic sequence of the genes that produce the isolated antibodies. Therecombinant DNA is then cloned and expressed in cells for large-scaleproduction of the newly humanized antibodies.

An example of a humanized antibody is a modified chimeric antibody. Achimeric antibody is generated as described above. The chimeric antibodyis further mutated outside of the CDRs to substitute non-human sequencesin the variable regions with the homologous human sequences. Anotherexample of a humanized antibody is a CDR-grafted antibody, in whichnon-human CDR sequences are introduced into the human heavy and lightchain variable sequences of a human antibody scaffold to replace thecorresponding human CDR sequences.

A humanized antibody can be produced in mammalian cells, bioreactors, ortransgenic animals, such as mice, chicken, sheep, goat, pig, andmarmoset. The transgenic animal can have a substantial portion of thehuman antibody-producing genome inserted into the genome of the animal.

A fully human monoclonal antibody corresponds to an antibody whoseantigen-binding residues are fully derived from the human immunoglobulinsequence or fragments thereof undergoing selection. In some embodiments,this selection can occur using phage display techniques in which aseries of variable antibody domain is expressed on a filamentous phagecoat protein and enriched for binding to a target antigen. In someembodiments, this selection can occur using transgenic animals, forexample, mice, rats, or rabbits, in which the entire set of endogenousimmunoglobulin genes are replaced with the entire set of humanimmunoglobulin genes. In some embodiments, the entire set of humanimmunoglobulin genes can be introduced into the genome of the animal andendogenous antibody production is rendered deficient in the productionof antibodies.

Humanized Monoclonal Antibodies that Target VE-PTP (HPTP-β)

The humanization process of a murine monoclonal antibody that targetsVE-PTP (HPTP-β) described herein utilizes a combination of CDR-graftingtechnologies coupled with antibody structure and a database of maturehuman IgG sequences. Human framework sequences were used as acceptorframeworks for the CDR sequences. These acceptor sequences alloriginated from mature Human IgG from a human source and not from phagedisplay.

Four humanized variants were designed for the heavy chain and lightchain variable domains of a murine monoclonal antibody raised againstthe recombinant extracellular domain of human VE-PTP (HPTP-β). Thedesigns were based on factors including homology, T-cell epitopes, keyresidues, and predicted structures.

The sequences disclosed herein were identified using antibody numberingsystems from IMGT and Kabat. These two numbering systems identifieddifferent residues of the murine antibodies as belonging to the CDR, anda combined IMGT/Kabat CDR sequence was used for optimal retention ofCDR-loop conformation.

Heavy Chain Sequences

SEQ ID NO: 1 is the V_(H) domain of a murine monoclonal antibody R15E6(V_(H0)) that targets human VE-PTP (HPTP-β), which includes the murinesignal peptide sequence (underlined). SEQ ID NO: 47 is a truncatedsequence of the V_(H) domain of R15E6 without the murine signal peptidesequence. SEQ ID NO: 2 is the closest human germline gene V-region, Homosapiens IGHV3-73, to the murine V_(H) domain of R15E6. SEQ ID NO: 3 andSEQ ID NO: 4 are Homo sapiens IGHV3-72 and Homo sapiens IGHV3-48,respectively, that are additional germline V-regions that are similar toSEQ ID NO: 2. The peptide sequences of SEQ ID NO: 1-4 are presented inTABLE 1.

TABLE 1 SEQ ID NO: Name Amino acid sequence  1 V_(H0)MDFGLSWVFFVVFYQGVHCEVQLVETGGGLVQ PKGSMKLSCAASGFTFNANAMNWIRQAPGKGLEWVARIRTKSNNYATYYAGSVKDRFTISRDDA QNMLYLQMNDLKTEDTAMYYCVRDYYGSSAWITYWGQGTLVTVSA 47 Truncated EVQLVETGGGLVQPKGSMKLSCAASGFTFNAN V_(H0)AMNWIRQAPGKGLEWVARIRTKSNNYATYYAG SVKDRFTISRDDAQNMLYLQMNDLKTEDTAMYYCVRDYYGSSAWITYWGQGTLVTVSA  2 IGHV3-73 EVQLVESGGGLVQPGGSLKLSCAASGFTFSGSAMHWVRQASGKGLEWVGRIRSKANSYATAYAA SVKGRFTISRDDSKNTAYLQMNSLKTEDTAVY YCTR 3 IGHV3-72 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDHYMDWVRQAPGKGLEWVGRTRNKANSYTTEYAA SVKGRFTISRDDSKNSLYLQMNSLKTEDTAVY YCAR 4 IGHV3-48 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTIYYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC AR

Online databases of Human IgG sequences were searched for comparison tothe murine V_(H) domain using BLAST search algorithms, and candidatehuman variable domains were selected from the top 200 BLAST results. Thecoordinate human variable domains were reduced to four candidates basedon a combination of framework homology, maintaining framework residues,and canonical loop structures. TABLE 2 lists the four selected acceptorframeworks, SEQ ID NO: 5-8. SEQ ID NO: 5 is AEX29600, SEQ ID NO: 6 isAAC51024, SEQ ID NO: 7 is AEX29289, and SEQ ID NO: 8 is ABA26204.

TABLE 2 SEQ ID NO: Name Amino acid sequence 5 AEX29600EVQLVESGGGLVQPGGSLKLSCAASGFTFSG SAMHWVRQASGKGLEWVGRIRSKANNYATAYAASVKGRFTISRDDSKNTAYLQMNSLKTEDT AAYYCIRDYYGATRGFQHWGQGTLVTVSS 6 AAC51024EVQLVESGGGLVQPGGSLRLSCAASGFTFSD HYMDWVRQAPGKGLEWVGRTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDT AVYYCARYVVGATLDYWGQGTLVTVSS 7 AEX29289EVQLVESGGGLVQPGRSLRLSCTASGFSFGD YAMNWVRQAPGKGLEWVGFIRSKTYGGTTEYAASVKGRFTISRDDSKNIAYLQMNSLKTEDT AVYYCTRDPADFYYYSSGQTGWFDPWGQGTL VTVSS 8ABA26204 LVQLVESGGGLVKPGGSLRLSCAASGFTFSD YYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV HYCARDGYSSSWYVDYWGQGTLVTVSS

Grafting the CDRs of the murine V_(H) into the acceptor frameworks (SEQID NO: 5-8) converted these sequences to humanized variants, which areshown in TABLE 3. SEQ ID NO: 9 is V_(H1), SEQ ID NO: 10 is V_(H2), SEQID NO: 11 is V_(H3), and SEQ ID NO: 12 is V_(H4).

TABLE 3 SEQ ID NO: Name Amino acid sequence  9 V_(H1)EVQLVESGGGLVQPGGSLKLSCAASGFTFNANAMNWVRQASGKGLEWVGRIRTKSNNYATYYAGSVKDRFTISRDDSKNTAYLQMNSLKTEDTAAYYCVRDYYGSSAWITYWGQ GTLVTVSS 10 V_(H2)EVQLVESGGGLVQPGGSLRLSCAASGFTFNANAMNWVRQAPGKGLEWVGRIRTKSNNYATYYAGSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRDYYGSSAWITYWGQ GTLVTVSS 11 V_(H3)EVQLVESGGGLVQPGRSLRLSCTASGFTFNANAMNWVRQAPGKGLEWVGRIRTKSNNYATYYAGSVKDRFTISRDDSKNIAYLQMNSLKTEDTAVYYCVRDYYGSSAWITYWGQ GTLVTVSS 12 V_(H4)LVQLVESGGGLVKPGGSLRLSCAASGFTFNANAMNWIRQAPGKGLEWVSRIRTKSNNYATYYAGSVKDRFTISRDNAKNSLYLQ1VINSLRAEDTAVHYCVRDYYGSSAWITYW GQGTLVTVSS

FIG. 1 shows a sequence alignment of the murine V_(H) sequence(truncated V_(H0); SEQ ID NO: 47) with four humanized variants SEQ IDNO: 9 (V_(H1)), SEQ ID NO: 10 (V_(H2)), SEQ ID NO: 11 (V_(H3)), and SEQID NO: 12 (V_(H4)). Residues important for the V_(H)/V_(K) interface andcanonical loop structure were preserved.

The percent homologies of the humanized variants to murine V_(H) arepresented in TABLE 4. The ranked order of homology of the humanizedvariants is V_(H2)>V_(H1)>V_(H3)>V_(H4).

TABLE 4 Humanized variant Identical amino acids Consensus amino acidsV_(H1) (SEQ ID NO: 9) 89.3% 94.3% V_(H2) (SEQ ID NO: 10) 90.2% 96.7%V_(H3) (SEQ ID NO: 11) 87.7% 95.1% V_(H4) (SEQ ID NO: 12) 86.9% 93.4%Light Chain Sequences

In TABLE 5 below, SEQ ID NO: 13 is the murine V_(L) domain of R15E6(V_(L0)), which includes the murine signal peptide sequence(underlined). SEQ ID NO: 48 is a truncated sequence of the V_(L) domainof R15E6 without the murine signal peptide sequence. SEQ ID NO: 14 isthe closest human germline gene V-region, Homo sapiens IGKV1-16, to themurine V_(L) domain of R15E6. SEQ ID NO: 15 is IGKV4-1 and is verysimilar to IGKV1-16.

TABLE 5 SEQ ID NO: Name Amino acid sequence 13 V_(L0)MESQTQVFVYMLLWLSGVEGDIVMTQSHKFMST SVGDRVSITCKASQHVGTAVAWYQQKPDQSPKQLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQ SEDLADYFCQQYSSYPFTFGSGTKLEIK 48Truncated DIVMTQSHKFMSTSVGDRVSITCKASQHVGTAV V_(L0)AWYQQKPDQSPKQLIYWASTRHTGVPDRFTGSG SGTDFTLTISNVQSEDLADYFCQQYSSYPFTFGSGTKLEIK 14 IGKV1-16 DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYNSYP 15IGKV4-1 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPD RFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYS TP

Online databases of Human IgK sequences were searched for comparison tothe murine V_(L) domain using BLAST search algorithms, and candidatehuman variable domains were selected from the top 200 BLAST results.These coordinate human variable domains were reduced to four candidatesbased on a combination of framework homology, maintaining frameworkresidues and canonical loop structure. TABLE 6 lists the four selectedacceptor frameworks. SEQ ID NO: 16 is AF234256_1, SEQ ID NO: 17 isAAD03722, SEQ ID NO: 18 is AAY33352, and SEQ ID NO: 19 is AAZ09113.

TABLE 6 SEQ ID NO: Name Amino acid sequence 15 AF234256_1DVVMTQSPSFLSASVGDRVTITCRASQGISNY LAWYQQRPGKAPKLLIYAASTLQTGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQLGGYPL TFGGGTKLEIK 17 AAD03722DIVMTQSPDSLAVSLGERATINCKSSQSVLY SSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYY CQQYYSTPYTFGQGTKLEIK 18 AAY33352DIQMTQSPFSLSASVGDRVTITCRASQGIGS SLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCLQHHD YPLTFGGGTKLEIK 19 AAZ09113DIVMTQSPDSLAVSLGERATINCKSSQSVFY SSNNKNYLAWYQQKPEQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYY CQQYYSSPLTFGGGTKVEIK

Grafting the CDRs of the murine V_(L) into these acceptor frameworksconverted these sequences to humanized variants, which are shown inTABLE 7. SEQ ID NO: 20 is V_(L1), SEQ ID NO: 21 is V_(L2), SEQ ID NO: 22is V_(L3), and SEQ ID NO: 23 is V_(L4).

TABLE 7 SEQ ID NO: Name Amino acid sequence 20 V_(L1)DVVMTQSPSFLSASVGDRVTITCKASQHVGTAVAWYQQRPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTIS SLQPEDFATYFCQQYSSYPFTFGGGTKLEIK21 V_(L2) DIVMTQSPDSLAVSLGERATINCKASQHVGTAVAWYQQKPGQPPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTIS SLQAEDVAVYYCQQYSSYPFTFGQGTKLEIK22 V_(L3) DIQMTQSPFSLSASVGDRVTITCKASQHVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTIS SLQPEDFATYFCQQYSSYPFTFGGGTKLEIK23 V_(L4) DIVMTQSPDSLAVSLGERATINCKASQHVGTAVAWYQQKPEQPPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTIS SLQAEDVAVYYCQQYSSYPFTFGGGTKVEIK

FIG. 2 shows a sequence alignment of the murine V_(L) sequence(truncated V_(L0); SEQ ID NO: 48) with four humanized V_(L) variants SEQID NO: 20 (V_(L1)), SEQ ID NO: 21 (V_(L2)), SEQ ID NO: 22 (V_(L3)), andSEQ ID NO: 23 (V_(L4)). Residues important for the V_(L)/V_(K) interfaceand canonical loop structure were preserved.

The percent homologies of the humanized variants to murine V_(L) arepresented in TABLE 8. The rank order of homology of the humanizedvariants is V_(L3)>V_(L1)>V_(L2)>V_(L4).

TABLE 8 Humanized variant Identical amino acids Consensus amino acidsV_(L1) (SEQ ID NO: 20) 81.3% 88.8% V_(L2) (SEQ ID NO: 21) 79.4% 88.8%V_(L3) (SEQ ID NO: 22) 82.2% 86.9% V_(L4) (SEQ ID NO: 23) 78.5% 89.7%Design of Humanized Antibodies: Combination of 4 V_(H) and 4 V_(L)Chains

Mouse variable chains were humanized by grafting the murine CDRsequences onto suitable human antibody donor sequences as describedabove in the context of the IgG4 heavy chain for the expression ofintact antibody proteins. A panel of full length humanized antibodieswas then codon-optimized for expression in the Chinese hamster ovary(CHO) cell line. The four variants designed for each variable chainusing different human donor sequences produced a matrix of 16 humanantibodies for expression, the heavy chain and light chain pairings ofwhich are shown in TABLE 9. A chimeric variant with full mouse variabledomains (V_(H0)-V_(L0)) grafted to human IgG4 constant domains wascreated as a positive control in binding and functional assays.

TABLE 9 V_(H1)-V_(L1) SEQ ID NO: 9-SEQ ID NO: 20 V_(H1)-V_(L2) SEQ IDNO: 9-SEQ ID NO: 21 V_(H1)-V_(L3) SEQ ID NO: 9-SEQ ID NO: 22V_(H1)-V_(L4) SEQ ID NO: 9-SEQ ID NO: 23 V_(H2)-V_(L1) SEQ ID NO: 10-SEQID NO: 20 V_(H2)-V_(L2) SEQ ID NO: 10-SEQ ID NO: 21 V_(H2)-V_(L3) SEQ IDNO: 10-SEQ ID NO: 22 V_(H2)-V_(L4) SEQ ID NO: 10-SEQ ID NO: 23V_(H3)-V_(L1) SEQ ID NO: 11-SEQ ID NO: 20 V_(H3)-V_(L2) SEQ ID NO:11-SEQ ID NO: 21 V_(H3)-V_(L3) SEQ ID NO: 11-SEQ ID NO: 22 V_(H3)-V_(L4)SEQ ID NO: 11-SEQ ID NO: 23 V_(H4)-V_(L1) SEQ ID NO: 12-SEQ ID NO: 20V_(H4)-V_(L2) SEQ ID NO: 12-SEQ ID NO: 21 V_(H4)-V_(L3) SEQ ID NO:12-SEQ ID NO: 22 V_(H4)-V_(L4) SEQ ID NO: 12-SEQ ID NO: 23T-Cell Epitope ScreenHeavy Chain.

The presentation of peptide sequences in the groove of MHC Class IImolecules leads to activation of CD8+ T-cells and an immunogenicresponse. To reduce this response, therapeutic proteins can be designedto avoid the incorporation of T-cell epitopes that can activate T-cellsby reducing the affinity of binding to the MHC Class II molecules.

The murine antibody V_(H) and V_(L) and the humanized variant sequenceswere screened for MHC II binding peptides to determine that thehumanization process had removed peptide sequences with high affinityusing in silico algorithms. TABLE 10 shows the results of the screen,with high affinity T-cell epitope cores in bold (IC₅₀<50 nM). The humangermline sequences ICHV3-73, ICHV3-72, and ICHV3-48 were also analyzedfor comparison. Any potential T-cell epitopes present in the germlinesequence and matched in the humanized variants are italicized. CDRs areunderlined.

TABLE 10 SEQ ID NO: Name Amino acid sequence  2 IGHV3-73EVQLVESGGGLVQPGGSLKLSCAASGFTFSGS AMHWVRQASGKGLEWVGRIRSKANSYATAYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVY YCTR  3 IGHV3-72EVQLVESGGGLVQPGGSLRLSCAASGFTFSDH YMDWVRQAPGKGLEWVGRTRNKANSYTTEYAASVKGRFTISRDDSKNSLYLQMNSLKTEDTAVY YCAR  4 IGHV3-48EVQLVESGGGLVQPGGSLRLSCAASGFTFSSY WMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC AR 47 TruncatedEVQLVETGGGLVQPKSMKLSCAASGFTFNANA V_(H0) MNWIRQAPGKGLEWVARIRTKSNNYATYYAGSVKDRFTISRDDAQNMLYLQMNDLKTEDTAMYY CVRDYYGSSAWITYWGQGTLVTVSA  9 V_(H1)EVQLVESGGGLVQPGGSLKLSCAASGFTFNAN AMNEVRQASGKGLEWVGRIRTKSNNYATYYAGSVKDRFTISRDDSKNTAYLQMNSLKTEDTAAY YCVRDYYGSSAWITYWGQGTLVTVSS 10 V_(H2)EVQLVESGGGLVQPGGSLRLSCAASGFTFNAN AMNWVRQAPGKGLEWVGRIRTKSNNYATYYAGSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVY YCVRDYYGSSAWITYWGQGTLVTVSS 11 V_(H3)EVQLVESGGGLVQPGRSLRLSCTASGFTFNAN AMNWVRQAPGKGLEWVGRIRTKSNNYATYYAGSVKDRFTISRDDSKNIAYLQMNSLKTEDTAVY YCVRDYYGSSAWITYWGQGTLVTVSS 12 V_(H4)LVQLVESGGGLVKPGGSLRLSCAASGFTFNAN AMNWIRQAPGKGLEWVSRIRTKSNNYATYYAGSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVY HYCVRDYYGSSAWITYWGQGTLVTVSS 54 HeavyGFTFNANAMN chain CDR1 55 Heavy RIRTKSNNYATYYAGSVKD chain CDR2 56 HeavyVRDYYGSSAWITY chain CDR3

TABLE 11 shows the results of the screen, with high affinity T-cellepitope cores in bold (IC₅₀<50 nM). The human germline sequencesIGKV1-16 and IGKV4-1 were also analyzed for comparison, and anypotential T-cell epitopes present in the germline sequence and matchedin the humanized variants, are italicized. The CDRs are underlined.

TABLE 11 SEQ ID NO: Name Amino acid sequence 14 IGKV1-16DIQMTQSPSSLSASVGDRVTITCRASQGISNYL AWFQQKPGKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP 15 IGKV4-1DIVQTQSPDLSAVSLGERATINCKSSQSVLYSS NNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYS TP 48 TruncatedDIVMTQSHKFMSTSVGDRVSITCKASQHVGTAV V_(L0)AWYQQKPDQSPKQLIYWASTRHTGVPDRFTGSG SGTDFTLTISNVQSEDLADYFCQQYSSYPFTFGSGTKLEIK 20 V_(L1) DVVMTQSPSFLSASVGDRVTITCKASQHVGTAVAWYQQRPGKAPKLLIYWASTRHTGVPSRFSGSG SGTEFTLTISSLQPEDFATYFCQQYSSYPFTFGGGTKLEIK 21 V_(L2) DIVMTQSPDSLAVSLGERATINCKASQHVGTAVAWYQQKPGQPPKLLIYWASTRHTGVPDRFSGSG SGTDFTLTISSLQAEDVAVYYCQQYSSYPFTFGQGTKLEIK 22 V_(L3) DIQMTQSPFSLSASVGDRVTITCKASQHVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSG SGTDFTLTISSLQPEDFATYFCQQYSSYPFTFGGGTKLEIK 23 V_(L4) DIVMTQSPDSLAVSLGERATINCKASQHVGTAVAWYQQKPEQPPKLLIYWASTRHTGVPDRFSGSG SGTDFTLTISSLQSEDVAVYYCQQYSSYPFTFGGGTKVEIK 57 Light KASQHVGTAVA chain CDR1 58 Light WASTRHT chain CDR2 59Light QQYSSYPFT chain CDR3

The murine V_(L0) contains a T-cell epitope within the CDR3/framework 4region of the light chain, which is not present in 3 of the humanizedvariants.

Post-Translational Modifications

Fv Glycosylation.

The N-linked glycosylation motif is NXS/T, where X is any amino acidexcept proline. This motif was not present in the murine or humanizedvariants of R15E6 V_(H) or V_(L).

Deamidation.

The amino acid motifs SNG, ENN, LNG, and LNN can be prone to deamidationof asparagine to provide aspartic acid. None of these four motifs werepresent in the murine or humanized variants of R15E6V_(H) or V_(L).

Signal Peptides.

Murine antibody signal peptides can result in higher levels ofexpression in CHO cells. The following signal peptides can beincorporated into the humanized variants.

Heavy chain signal peptide (SEQ ID NO: 24): MGWTLVFLFLLSVTAGVHSLight chain signal peptide (SEQ ID NO: 25): MVSSAQFLGLLLLCFQGTRC

Each of the V_(H) domains can be synthesized in-frame with a human IgG4isotype constant domain sequence, with a stabilizing S228P mutation. Theentire heavy chain sequence can be codon optimized, and the DNA sequencecan be verified. The amino acid sequence of the IgG4 constant domainwith S228P mutation (SEQ ID NO: 26) is:

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Each of the V_(L) domains can be synthesized in-frame with a human IgKisotype constant domain sequence. The entire light chain sequence canthen be codon optimized, and the DNA sequence can be verified. The aminoacid sequence of the IgK constant domain (SEQ ID NO: 27) is:

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

Each of the variant chains can be verified by DNA sequencing analysis.Then, transient transfection and expression of each of the humanizedantibodies can be pursued. One chimeric antibody can be expressed foruse as a positive control and can have the murine variable domains, thehuman Ig constant domains. The 16 humanized variants only have humanizedvariable domains and human Ig constant domains. TABLE 12 shows the heavychain and light chain parings that provide the 16 humanized variantswith humanized variable domains and human Ig constant domains.

TABLE 12 Chimeric Antibody HC0-LC0 Humanized Variants HC1-LC1 HC1-LC2HC1-LC3 HC1-LC4 HC2-LC1 HC2-LC2 HC2-LC3 HC2-LC4 HC3-LC1 HC3-LC2 HC3-LC3HC3-LC4 HC4-LC1 HC4-LC2 HC4-LC3 HC4-LC4

TABLE 13 and TABLE 14 list the full amino acid sequences of eachhumanized heavy and light chain.

TABLE 13 SEQ ID NO: Name Amino acid sequence 28 HC0MGWTLVFLFLLSVTAGVHSEVQLVETGGGLVQPKGSMKLSCAASGFTENANAMNWIRQAPGKGLEWVARIRTKSNNYATYYAGSVKDRFTISRDDAQNMLYLQMNDLKTEDTAMYYCVRDYYGSSAWITWGQGTLVTVSAASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDKSRWQEGNVFS CSVMHEALHNHYTQKSLSLSLGK 29 HC1MGWTLVFLFLLSVTAGVHSEVQLVESGGGLVQPGGSLKLSCAASGFTFNANAMNWVRQASGKGLEWVGRIRTKSNNYATYYAGSVKDRFTISRDDSKNTAYLQMNSLKTEDTAAYYCVRDYYGSSAWITYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK 30 HC2MGWTLVFLFLLSVTAGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFNANAMNWVRQAPGKGLEWVGRIRTKSNNYATYYAGSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRDYYGSSAWITYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP55SLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK 31 HC3MGWTLVFLFLLSVTAGVHSEVQLVESGGGLVQPGRSLRLSCTASGFTFNANAMNWVRQAPGKGLEWVGRIRTKSNNYATYYAGSVKDRFTISRDDSKNIAYLQMNSLKTEDTAVYYCVRDYYGSSAWITYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK 32 HC4MGWTLVFLFLLSVTAGVHSLVQLVESGGGLVKPGGSLRLSCAASGFTENANAMNWIRQAPGKGLEWVSRIRTKSNNYATYYAGSVKDRFTISRDNAKNSLYLQMNSLRAEDTAVHYCVRDYYGSSAWITYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSLGK

TABLE 14 SEQ ID NO: Name Amino acid sequence 33 LC0MVSSAQFLGLLLLCFQGTRCDIVMTQSHKFMSTSVGDRVSITCKASQHVGTAVAWYQQKPDQSPKQLIYWASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCQQYSSYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 34 LC1MVSSAQFLGLLLLCFQGTRCDVVMTQSPSFLSASVGDRVTITCKASQHVGTAVAWYQQRPGKAPKLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQYSSYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 35 LC2MVSSAQFLGLLLLCFQGTRCDIVMTQSPDSLAVSLGERATINCKASQHVGTAVAWYQQKPGQPPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSSYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 36 LC3MVSSAQFLGLLLLCFQGTRCDIQMTQSPFSLSASVGDRVTITCKASQHVGTAVAWYQQKPGKAPKLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYSSYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 37 LC4MVSSAQFLGLLLLCFQGTRCDIVMTQSPDSLAVSLGERATINCKASQHVGTAVAWYQQKPEQPPKLLIYWASTRHTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYSSYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

Target sequences can have at least about 80% homology, at least about81% homology, at least about 82% homology, at least about 83% homology,at least about 84% homology, at least about 85% homology, at least about86% homology, at least about 87% homology, at least about 88% homology,at least about 89% homology, at least about 90% homology, at least about91% homology, at least about 92% homology, at least about 93% homology,at least about 94% homology, at least about 95% homology, at least about96% homology, at least about 97% homology, at least about 98% homology,at least about 99% homology, at least about 99.1% homology, at leastabout 99.2% homology, at least about 99.3% homology, at least about99.4% homology, at least about 99.5% homology, at least about 99.6%homology, at least about 99.7% homology, at least about 99.8% homology,at least about 99.9% homology, at least about 99.91% homology, at leastabout 99.92% homology, at least about 99.93% homology, at least about99.94% homology, at least about 99.95% homology, at least about 99.96%homology, at least about 99.97% homology, at least about 99.98%homology, or at least about 99.99% homology to an amino acid sequenceprovided herein.

Various methods and software programs can be used to determine thehomology between two or more peptides or nucleic acids, such as NCBIBLAST, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, or anothersuitable method or algorithm.

Pharmaceutical Compositions

A pharmaceutical composition of the invention can be a combination ofany pharmaceutical compounds described herein with other chemicalcomponents, such as carriers, stabilizers, diluents, dispersing agents,suspending agents, thickening agents, and/or excipients. Thepharmaceutical composition facilitates administration of the compound toan organism. Pharmaceutical compositions can be administered intherapeutically-effective amounts as pharmaceutical compositions byvarious forms and routes including, for example, intravenous,intramuscular, oral, parenteral, ophthalmic, and topical administration.

A pharmaceutical composition can be administered to the eye via anysuitable form or route including, for example, topical, oral, systemic,intravitreal, intracameral, subconjunctival, subtenon, retrobulbar,intraocular, posterior juxtascleral, periocular, subretinal, andsuprachoroidal administration. The compositions can be administered byinjecting the formulation in any part of the eye including anteriorchamber, posterior chamber, vitreous chamber (intravitreal), retinaproper, and/or subretinal space. The compositions can be delivered via anon-invasive method. Non-invasive modes of administering the formulationcan include using a needleless injection device. Multiple administrationroutes can be employed for efficient delivery of the pharmaceuticalcompositions.

A pharmaceutical composition can be targeted to any suitable ocular cellincluding, for example, endothelial cells such as vascular endothelialcells, cells of the retina such as retinal pigment epithelium (RPE),corneal cells, fibroblasts, astrocytes, glial cells, pericytes, irisepithelial cells, cells of neural origin, ciliary epithelial cells,Müller cells, muscle cells surrounding and attached to the eye such ascells of the lateral rectus muscle, orbital fat cells, cells of thesclera and episclera, cells of the trabecular meshwork, and connectivetissue cells.

A pharmaceutical composition can be administered in a local manner, forexample, via injection of the compound directly into an organ,optionally in a depot or sustained release formulation or implant.Pharmaceutical compositions can be provided in the form of a rapidrelease formulation, in the form of an extended release formulation, orin the form of an intermediate release formulation. A rapid release formcan provide an immediate release. An extended release formulation canprovide a controlled release or a sustained delayed release.

Pharmaceutical formulations for administration can include aqueoussolutions of the active compounds in water-soluble form. Suspensions ofthe active compounds can be prepared as oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acid esters, such as ethyl oleate ortriglycerides, or liposomes. Aqueous injection suspensions can containsubstances which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol, or dextran. The suspension canalso contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions. Alternatively, the active ingredient can be inpowder form for constitution with a suitable vehicle, for example,sterile pyrogen-free water, before use.

In practicing the methods of treatment or use provided herein,therapeutically-effective amounts of the compounds described herein areadministered in pharmaceutical compositions to a subject having adisease or condition to be treated. In some embodiments, the subject isa mammal such as a human. A therapeutically-effective amount can varywidely depending on the severity of the disease, the age and relativehealth of the subject, the potency of the compounds used, and otherfactors.

In some embodiments, the compounds described herein can be used singlyor in combination with one or more therapeutic agents as components ofmixtures. For example, a VE-PTP (HPTP-β) inhibitor of the disclosure canbe co-formulated or co-administered with antibodies, for example,anti-VEGF agents. An anti-VEGF agent can be a compound, an antibody, oran antibody fragment, variant, or derivative thereof. Non-limitingexamples of an anti-VEGF agent include bevacizumab (Avastin®),ranibizumab (Lucentis®), and aflibercept (Eylea®). In some embodiments,the compounds described herein can be used before, during, or aftertreatment with an anti-VEGF agent.

Pharmaceutical compositions can be formulated using one or morephysiologically-acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active compounds intopreparations that can be used pharmaceutically. Formulation can bemodified depending upon the route of administration chosen.Pharmaceutical compositions comprising compounds described herein can bemanufactured, for example, by mixing, dissolving, emulsifying,encapsulating, entrapping, or compression processes.

The pharmaceutical compositions can include at least onepharmaceutically-acceptable carrier, diluent, or excipient and compoundsdescribed herein as free-base or pharmaceutically-acceptable salt form.Pharmaceutical compositions can contain solubilizers, stabilizers,tonicity enhancing agents, buffers, and preservatives.

Methods for the preparation of compositions comprising the compoundsdescribed herein include formulating the compounds with one or moreinert, pharmaceutically-acceptable excipients or carriers to form asolid, semi-solid, or liquid composition. Solid compositions include,for example, powders, tablets, dispersible granules, capsules, andcachets. Liquid compositions include, for example, solutions in which acompound is dissolved, emulsions comprising a compound, or a solutioncontaining liposomes, micelles, or nanoparticles comprising a compoundas disclosed herein. Semi-solid compositions include, for example, gels,suspensions and creams. The compositions can be in liquid solutions orsuspensions, solid forms suitable for solution or suspension in a liquidprior to use, or as emulsions. These compositions can also contain minoramounts of nontoxic, auxiliary substances, such as wetting oremulsifying agents, pH buffering agents, and otherpharmaceutically-acceptable additives.

Non-limiting examples of dosage forms suitable for use in the inventioninclude liquid, powder, gel, nanosuspension, nanoparticle, microgel,aqueous or oily suspensions, emulsion, and any combination thereof.

Non-limiting examples of pharmaceutically-acceptable excipients suitablefor use in the invention include binding agents, disintegrating agents,anti-adherents, anti-static agents, surfactants, anti-oxidants, coatingagents, coloring agents, plasticizers, preservatives, suspending agents,emulsifying agents, anti-microbial agents, spheronization agents, andany combination thereof.

A composition of the invention can be, for example, an immediate releaseform or a controlled release formulation. An immediate releaseformulation can be formulated to allow the compounds to act rapidly.Non-limiting examples of immediate release formulations include readilydissolvable formulations. A controlled release formulation can be apharmaceutical formulation that has been adapted such that release ratesand release profiles of the active agent can be matched to physiologicaland chronotherapeutic requirements or, alternatively, has beenformulated to effect release of an active agent at a programmed rate.Non-limiting examples of controlled release formulations includegranules, delayed release granules, hydrogels (e.g., of synthetic ornatural origin), other gelling agents (e.g., gel-forming dietaryfibers), matrix-based formulations (e.g., formulations comprising apolymeric material having at least one active ingredient dispersedthrough), granules within a matrix, polymeric mixtures, and granularmasses.

In some, a controlled release formulation is a delayed release form. Adelayed release form can be formulated to delay a compound's action foran extended period of time. A delayed release form can be formulated todelay the release of an effective dose of one or more compounds, forexample, for about 4, about 8, about 12, about 16, or about 24 hours.

A controlled release formulation can be a sustained release form. Asustained release form can be formulated to sustain, for example, thecompound's action over an extended period of time. A sustained releaseform can be formulated to provide an effective dose of any compounddescribed herein (e.g., provide a physiologically-effective bloodprofile) over about 4, about 8, about 12, about 16, or about 24 hours.

The disclosed compositions can optionally comprisepharmaceutically-acceptable preservatives.

Non-limiting examples of pharmaceutically-acceptable excipients can befound, for example, in Remington: The Science and Practice of Pharmacy,Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, JohnE., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999), each of which is incorporated by reference in itsentirety.

A compound described herein can be conveniently formulated intopharmaceutical compositions composed of one or morepharmaceutically-acceptable carriers. See e.g., Remington'sPharmaceutical Sciences, latest edition, by E. W. Martin Mack Pub. Co.,Easton, Pa., incorporated by reference in its entirety, which disclosestypical carriers and conventional methods of preparing pharmaceuticalcompositions. Such carriers can be carriers for administration ofcompositions to humans and non-humans, including solutions such assterile water, saline, and buffered solutions at physiological pH.Pharmaceutical compositions can also include one or more additionalactive ingredients such as antimicrobial agents, anti-inflammatoryagents, and anesthetics.

Non-limiting examples of pharmaceutically-acceptable carriers includesaline, Ringer's solution, and dextrose solution. In some embodiments,the pH of the solution can be from about 5 to about 8, and can be fromabout 7 to about 7.5. Further carriers include sustained releasepreparations such as semipermeable matrices of solid hydrophobicpolymers containing the compound. The matrices can be in the form ofshaped articles, for example, films, liposomes, microparticles, ormicrocapsules.

The disclosed methods relate to administering an antibody targetingVE-PTP (HPTP-β) as part of a pharmaceutical composition. Compositionssuitable for topical administration can be used. In some embodiments,compositions of the invention can comprise a liquid comprising an activeagent in solution, in suspension, or both. Liquid compositions caninclude gels. A liquid composition can be, for example, aqueous. Acomposition is an in situ gellable aqueous composition. In iteration,the composition is an in situ gellable aqueous solution. Such acomposition can comprise a gelling agent in a concentration effective topromote gelling upon contact with the eye or lacrimal fluid in theexterior of the eye. Aqueous compositions can haveophthalmically-compatible pH and osmolality. The composition cancomprise an ophthalmic depot formulation comprising an active agent forsubconjunctival administration. Microparticles comprising an activeagent can be embedded in a biocompatible, pharmaceutically-acceptablepolymer or a lipid encapsulating agent. The depot formulations can beadapted to release all or substantially all the active material over anextended period of time. The polymer or lipid matrix, if present, can beadapted to degrade sufficiently to be transported from the site ofadministration after release of all or substantially all the activeagent. The depot formulation can be a liquid formulation, comprising apharmaceutical acceptable polymer and a dissolved or dispersed activeagent. Upon injection, the polymer forms a depot at the injections site,for example, by gelifying or precipitating. The composition can comprisea solid article that can be inserted in a suitable location in the eye,such as between the eye and eyelid or in the conjunctival sac, where thearticle releases the active agent. Solid articles suitable forimplantation in the eye in such fashion can comprise polymers and can bebioerodible or non-bioerodible.

Pharmaceutical formulations can include additional carriers, as well asthickeners, diluents, buffers, preservatives, and surface active agentsin addition to the agents disclosed herein.

The pH of the disclosed composition can range from about 3 to about 12.The pH of the composition can be, for example, from about 3 to about 4,from about 4 to about 5, from about 5 to about 6, from about 6 to about7, from about 7 to about 8, from about 8 to about 9, from about 9 toabout 10, from about 10 to about 11, or from about 11 to about 12 pHunits. The pH of the composition can be, for example, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 11, orabout 12 pH units. The pH of the composition can be, for example, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 11 or at least 12 pH units. The pH of thecomposition can be, for example, at most 3, at most 4, at most 5, atmost 6, at most 7, at most 8, at most 9, at most 10, at most 11, or atmost 12 pH units. If the pH is outside the range desired by theformulator, the pH can be adjusted by using sufficientpharmaceutically-acceptable acids and bases.

Depending on the intended mode of administration, the pharmaceuticalcompositions can be in the form of solid, semi-solid or liquid dosageforms, such as, for example, tablets, suppositories, pills, capsules,powders, liquids, suspensions, lotions, creams, or gels, for example, inunit dosage form suitable for single administration of a precise dosage.

For solid compositions, nontoxic solid carriers include, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talc, cellulose, glucose, sucrose, and magnesiumcarbonate.

Non-limiting examples of pharmaceutically active agents suitable forcombination with compositions of the disclosure include anti-infectives,i.e., aminoglycosides, antiviral agents, antimicrobials,anti-cholinergics/anti-spasmodics, antidiabetic agents, antihypertensiveagents, anti-neoplastics, cardiovascular agents, central nervous systemagents, coagulation modifiers, hormones, immunologic agents,immunosuppressive agents, and ophthalmic preparations.

In some embodiments, the pharmaceutical composition provided hereincomprises a therapeutically effective amount of a compound in admixturewith a pharmaceutically-acceptable carrier and/or excipient, forexample, saline, phosphate buffered saline, phosphate and amino acids,polymers, polyols, sugar, buffers, preservatives, and other proteins.Illustrative agents include octylphenoxy polyethoxy ethanol compounds,polyethylene glycol monostearate compounds, polyoxyethylene sorbitanfatty acid esters, sucrose, fructose, dextrose, maltose, glucose,mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose,trehalose, bovine or human serum albumin, citrate, acetate, Ringer's andHank's solutions, cysteine, arginine, carnitine, alanine, glycine,lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycol.

Methods of Administration and Treatment Methods

Pharmaceutical compositions described herein can be administered forprophylactic and/or therapeutic treatments. In therapeutic applications,the compositions can be administered to a subject already suffering froma disease or condition, in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease or condition, or to cure,heal, improve, or ameliorate the condition. Compositions can also beadministered to lessen a likelihood of developing, contracting, orworsening a condition. Amounts effective for this use can vary based onthe severity and course of the disease or condition, previous therapy,the subject's health status, weight, and response to the drugs, and thejudgment of the treating physician.

Multiple therapeutic agents can be administered in any order orsimultaneously. If simultaneously, the multiple therapeutic agents canbe provided in a single, unified form, or in multiple forms, forexample, as multiple separate pills. The agents can be packed togetheror separately, in a single package or in a plurality of packages. One orall of the therapeutic agents can be given in multiple doses. If notsimultaneous, the timing between the multiple doses can vary to as muchas about a month.

Therapeutic agents described herein can be administered before, during,or after the occurrence of a disease or condition, and the timing ofadministering the composition containing a therapeutic agent can vary.For example, the compositions can be used as a prophylactic and can beadministered continuously to subjects with a propensity to conditions ordiseases in order to lessen a likelihood of the occurrence of thedisease or condition. The compositions can be administered to a subjectduring or as soon as possible after the onset of the symptoms. Theadministration of the therapeutic agents can be initiated within thefirst 48 hours of the onset of the symptoms, within the first 24 hoursof the onset of the symptoms, within the first 6 hours of the onset ofthe symptoms, or within 3 hours of the onset of the symptoms. Theinitial administration can be via any practical route, such as by anyroute described herein using any formulation described herein. Atherapeutic agent can be administered as soon as is practicable afterthe onset of a disease or condition is detected or suspected, and for alength of time necessary for the treatment of the disease, such as, forexample, from about 1 month to about 3 months. The length of treatmentcan vary for each subject.

Pharmaceutical compositions described herein can be in unit dosage formssuitable for single administration of precise dosages. In unit dosageform, the formulation is divided into unit doses containing appropriatequantities of one or more compounds. The unit dosage can be in the formof a package containing discrete quantities of the formulation.Non-limiting examples are packaged injectables, vials, or ampoules.Aqueous suspension compositions can be packaged in single-dosenon-reclosable containers. Multiple-dose reclosable containers can beused, for example, in combination with or without a preservative.Formulations for injection can be presented in unit dosage form, forexample, in ampoules, or in multi-dose containers with a preservative.

Pharmaceutical compositions provided herein can be administered inconjunction with other therapies, for example, chemotherapy, radiation,surgery, anti-inflammatory agents, and selected vitamins. The otheragents can be administered prior to, after, or concomitantly with thepharmaceutical compositions.

Dosing

The VE-PTP (HPTP-β) antibody can be administered at a dosage of about0.01 mg/kg to about 50 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 1mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 1 mg/kgto about 5 mg/kg, or about 3 mg/kg to about 7 mg/kg by weight of thesubject.

The VE-PTP (HPTP-β) antibody can be administered at any intervaldesired. The administration of the compound can have irregular dosingschedules to accommodate either the person administering the compound orthe subject receiving the compound. For example, the compound can beadministered once a week, 2 times a week, 3 times a week, 4 times aweek, 6 times a week, 6 times a week, 7 times a week, 8 times a week, 9times a week or 10 times a week. The interval between daily dosing canbe any hourly interval, for example, every hour, every 2 hours, every 3hours, every 4 hours, every 5 hours, every 6 hours, every 7 hours, every8 hours, every 9 hours, every 10 hours, every 11 hours, or every 12hours. The dosing schedules for administration of a VE-PTP (HPTP-β)antibody include, but are not limited to, once daily, three-timesweekly, twice weekly, once weekly, three times, monthly, twice monthly,once monthly, once every other month, yearly, two-timed yearly,three-times yearly, or four-times yearly. In some embodiments, theVE-PTP (HPTP-β) antibody can be administered every other week.

In addition, the amount of the VE-PTP (HPTP-β) antibody administered canbe of the same amount in each dose or the dosage can vary between doses.For example, a first amount dosed in the morning and a second amountadministered in the evening. The dosage for administration can varydepending upon the schedule of the anti-VEGF administration.

The anti-VE-PTP antibody can be administered in combination with anyanti-VEGF agent in any combination, for example, at the beginning of thetreatment, at any time during the treatment or at any time aftertreatment with the anti-VEGF agent has concluded. In addition, thedosage of the VE-PTP (HPTP-β) inhibitors can be adjusted duringtreatment. Also, the amount of anti-VEGF agent can be adjusted duringtreatment.

Further non-limiting examples of VEGF-modulating agents includenon-inflammatory agents, for example, dexamethasone, fluocinolone, andtriamcinolone. In addition, the disclosed methods can include implantswhich deliver an anti-VEGF agent. For example, VE-PTP (HPTP-β)inhibitors can be co-administered either before, during or after animplant is provided to a subject suffering from a disease or conditiondescribed herein.

Anti-VEGF treatments can be administered, for example, monthly, onceevery 3 months, once every 6 months, or yearly, wherein the VE-PTP(HPTP-β) inhibitor is administered at any frequency between treatments.

Also disclosed herein are methods for treating a disease or condition asdisclosed herein. The method comprises administering to a subject:

a) a therapeutically-effective amount of a VE-PTP (HPTP-β) inhibitor;and

b) a therapeutically-effective amount of an anti-VEGF agent;

wherein the administration of the VE-PTP (HPTP-β) inhibitor and theanti-VEGF agent can be conducted in any manner desired by theadministrator, for example, as further described herein.

A VE-PTP (HPTP-β) binding agent or an anti-VEGF agent can beadministered in any amount necessary or convenient. For example, acompound described herein can be administered in an amount from about0.1 mg to about 300 mg, about 0.1 mg to about 200 mg, about 0.1 mg toabout 100 mg, about 0.05 mg to about 1.5 mg, 0.1 mg to about 1.5 mg,about 0.05 mg to about 1 mg, or about 0.1 mg to about 1 mg, about 0.05mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about0.1 mg, about 0.11 mg, about 0.12 mg, about 0.13 mg, about 0.14 mg,about 0.15 mg, about 0.16 mg, about 0.17, mg, about 0.18 mg, about 0.19mg, about 0.2 mg, about 0.21 mg, about 0.22 mg, about 0.23 mg, about0.24 mg, about 0.25 mg, about 0.26 mg, about 0.27, mg, about 0.28 mg,about 0.29 mg, about 0.3 mg, about 0.31 mg, about 0.32 mg, about 0.33mg, about 0.34 mg, about 0.35 mg, about 0.36 mg, about 0.37, mg, about0.38 mg, about 0.39 mg, about 0.4 mg, about 0.41 mg, about 0.42 mg,about 0.43 mg, about 0.44 mg, about 0.45 mg, about 0.46 mg, about 0.47,mg, about 0.48 mg, about 0.49 mg, about 0.5 mg, about 0.51 mg, about0.52 mg, about 0.53 mg, about 0.54 mg, about 0.55 mg, about 0.56 mg,about 0.57, mg, about 0.58 mg, about 0.59 mg, about 0.6 mg, about 0.61mg, about 0.62 mg, about 0.63 mg, about 0.64 mg, about 0.65 mg, about0.66 mg, about 0.67, mg, about 0.68 mg, about 0.69 mg, about 0.7 mg,about 0.71 mg, about 0.72 mg, about 0.73 mg, about 0.74 mg, about 0.75mg, about 0.76 mg, about 0.77, mg, about 0.78 mg, about 0.79 mg, about0.8 mg, about 0.81 mg, about 0.82 mg, about 0.83 mg, about 0.84 mg,about 0.85 mg, about 0.86 mg, about 0.87, mg, about 0.88 mg, about 0.89mg, about 0.9 mg, about 0.91 mg, about 0.92 mg, about 0.93 mg, about0.94 mg, about 0.95 mg, about 0.96 mg, about 0.97, mg, about 0.98 mg,about 0.99 mg, about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg,about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about8.5 mg, about 9 mg, about 9.5 mg, about 10 mg, about 15 mg, about 20 mg,about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg,about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about160 mg, about 170 mg, about 180 mg, about 190 mg about 200 mg, about 210mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260mg, about 270 mg, about 280 mg, about 290 mg, or about 300 mg per dosefor a subject by any route of administration.

Intraocular Delivery

Disclosed herein are methods for intraocular delivery of compositions ofthe invention to a subject having a disease or condition as disclosedherein. The delivery method can include an invasive method for directdelivery of the composition to ocular cells. In some embodiments, aliquid pharmaceutical composition comprising the antibody is deliveredvia a subretinal injection. In some embodiments, a liquid pharmaceuticalcomposition comprising the antibody is delivered via intravitreal orsubcutaneous injection. In some embodiments, a liquid pharmaceuticalcomposition comprising the antibody is delivered via intracameralinjection. In some embodiments, the composition is delivered viamultiple administration routes, for example, subretinal and/orintravitreous, to increase efficiency of the antibody delivery. In someembodiments, the subretinal and/or intravitreal injection is preceded bya vitrectomy.

The intraocular injection can be performed over any interval of time toimprove efficiency of delivery and/or to minimize or avoid damage tosurrounding tissue. The interval of time for the intraocular injectioncan be from, for example, about 1 minute to about 60 minutes, about 1minute to about 5 minutes, about 5 minutes to about 10 minutes, about 10minutes to about 15 minutes, about 15 minutes to about 20 minutes, about20 minutes to about 25 minutes, about 25 minutes to about 30 minutes,about 30 minutes to about 35 minutes, about 35 minutes to about 40minutes, about 40 minutes to about 45 minutes, about 45 minutes to about50 minutes, about 50 minutes to about 55 minutes, or about 55 minutes toabout 60 minutes.

The intraocular injection can be performed at any rate. The rate ofintraocular injection can be from, for example, about 1 μL/min to about200 μL/min, about 1 μL/min to about 10 μL/min, about 10 μL/min to about20 μL/min, about 20 μL/min to about 30 μL/min, about 30 μL/min to about40 μL/min, about 40 μL/min to about 50 μL/min, about 50 μL/min to about60 μL/min, about 60 μL/min to about 70 μL/min, about 70 μL/min to about80 μL/min, about 80 μL/min to about 90 μL/min, about 90 μL/min to about100 μL/min, about 100 μL/min to about 110 μL/min, about 110 μL/min toabout 120 μL/min, about 120 μL/min to about 130 μL/min, about 130 μL/minto about 140 μL/min, about 140 μL/min to about 150 μL/min, about 150μL/min to about 160 μL/min, about 160 μL/min to about 170 μL/min, about170 μL/min to about 180 μL/min, about 180 μL/min to about 190 μL/min, orabout 190 μL/min to about 200 μL/min.

Kits

The present disclosure further relates to kits containing thecomposition of the disclosure. A kit can comprise:

A) a composition comprising an antibody targeting VE-PTP (HPTP-β); and

B) a carrier for delivering the composition to a subject.

The kits can be modified to fit the dosing regimen prescribed for thesubject being treated. The following is a non-limiting example of a kitfor use with a subject receiving a composition of the disclosure by anintraocular injection:

A) an aqueous composition containing:

-   -   a) an antibody targeting the VE-PTP (HPTP-β) extracellular        domain; and    -   b) a carrier system, comprising:        -   i) a tonicity agent; and        -   ii) water        -   wherein the tonicity agent is present in an amount such that            the such that the re-constituted formula comprises from            about 0.5% to about 10% mass to volume of the tonicity            agent; and

B) a component for delivering the aqueous composition.

In some embodiments, a kit of the invention comprises:

A) a composition for delivering an anti-VE-PTP antibody; and

B) a composition for delivering an anti-VEGF agent.

The kits can be modified to fit the dosing regimen prescribed for thesubject being treated. The following is a non-limiting example of a kitfor use with a patient receiving an intravenously-delivered compositioncomprising the disclosed compounds and an intravitreally-administeredanti-VEGF agent. This example provides dosing of the disclosed compoundstwice daily for 3 months and for an injection of ranibizumab at week 12.

A. 3 packages, each package containing 4 vials. Each vial comprising asufficient amount of a VE-PTP (HPTP-β) inhibitor to provide 2 dailyinjections of 5 mg of the disclosed compounds for 7 days; and

B. a vial of ranibizumab for injection at the end of week 12 whichprovides 0.5 mg of ranibizumab.

Kits can comprise any combination of elements. In addition, when ananti-VE-PTP antibody disclosed herein is provided orally, a singlecontainer with sufficient doses of the disclosed compounds can besupplied with the kit.

A kit can also provide written instructions for use and disposal of thecompositions to be delivered. The instructions can be modified from kitto kit to reflect the dosing regimen prescribed. The instructions candescribe any therapy, compounds, excipients, or method of administrationdescribed herein.

The disclosed compositions can comprise, for example, from about 1.5% toabout 90% mass by volume of a carrier system. Non-limiting examples oftonicity agents include dextrose, mannitol, and glycerin. The formulatorcan utilize more than one tonicity agent.

The kit can further comprise devices for administration, such as asyringe, filter needle, extension tubing, cannula, and subretinalinjector. Non-limiting examples of routes of administration includeintraocular, parenteral, and topical. Intraocular routes ofadministration can include, for example, intravitreal, intracameral,subconjunctival, subtenon, retrobulbar, intraocular, posteriorjuxtascleral, periocular, subretinal, and suprachoroidal. Delivery canbe by, for example, syringe, needle, infusion pump, or injector.Syringes and injectors can be, for example, single-dose, multi-dose,fixed-dose, or variable-dose. Non-limiting examples of injectorsinclude, pen injectors, auto-injectors, and electronic patch injectorsystems.

The kits can comprise suitable components for the administration of acomposition of the invention to a subject. In some embodiments acomposition of the invention is present in the kit as a unit dosageform. As such, the formulator can provide delivery devices having ahigher concentration of compound and adjust the delivered volume toprovide an amount of compound that is less than the amount in the entiresolution.

A set of instructions can be included in any kit described herein. Theinstructions can relate to the dosing amount, timing of dosing,reconstitution of the composition when the kit contains a drycomposition, and methods of disposal of delivery vehicles and unusedcomposition. The instructions can describe any therapy, compounds,excipients, or method of administration described herein.

Methods

The invention provides compositions and methods for the treatment orprevention of diseases or conditions of the eye, for example, ocularedema, diabetic macular edema, vascular leak, age-related maculardegeneration (wet form), age-related macular degeneration (dry form),choroidal neovascularization, diabetic retinopathy, ocular ischemia,uveitis, retinal vein occlusion (central or branch), ocular trauma,surgery induced edema, surgery induced neovascularization, cystoidmacular edema, ocular ischemia, and uveitis. These diseases orconditions can be characterized by changes in the ocular vasculature,whether progressive or non-progressive, whether a result of an acutedisease or condition, or a chronic disease or condition. These diseasescan be characterized by an increased level of plasma vascularendothelial growth factor (VEGF).

The disclosure provides a method of treating ocular neovascularizationin a subject in need thereof, the method comprising administering to thesubject a therapeutically-effective amount of an antibody targetingVE-PTP (HPTP-β).

In some embodiments, the VE-PTP (HPTP-β) inhibitor stabilizes thevasculature against leakage and neovascularization.

Improvement of clinical symptoms can be monitored, for example, byindirect ophthalmoscopy, fundus photography, fluorescein angiopathy,electroretinography, external eye examination, slit lamp biomicroscopy,applanation tonometry, pachymetry, optical coherence tomography, orautorefraction. In some embodiments, the disclosed methods relate to theadministration of the VE-PTP (HPTP-β) antibody, including compositionscomprising an anti-VEGF agent.

In some embodiments, the methods of the disclosure includeco-administration of a VE-PTP (HPTP-β) antibody with one or moreanti-VEGF agents, which can stabilize the vasculature against leakage.

In some embodiments, the methods of the disclosure are drawn towardsco-administration of an anti-VE-PTP (HPTP-β) antibody with one or moreanti-VEGF agents, which can stabilize the vasculature againstneovascularization.

In some embodiments, the anti-VE-PTP (HPTP-β) antibody can stabilize thevasculature against leakage and neovascularization.

In some embodiments, a human subject with at least one visually-impairedeye is treated with from about 0.1 mg to about 100 mg of a VE-PTP(HPTP-β) antibody. Improvement of clinical symptoms can be monitored by,for example, indirect ophthalmoscopy, fundus photography, fluoresceinangiopathy, electroretinography, external eye examination, slit lampbiomicroscopy, applanation tonometry, pachymetry, optical coherencetomography and autorefraction. As described herein, the dosing can occurat any frequency determined by the administrator. After cessation of theanti-VEGF agent treatment, subsequent doses can be administered, forexample, weekly or monthly, e.g., with a frequency of about 2-8 weeks orabout 1-12 months apart depending upon the response.

Diseases that are a direct or indirect result of diabetes include, interalia, diabetic macular edema and diabetic retinopathy. The ocularvasculature of the diabetic becomes unstable over time and leads toconditions such as non-proliferative retinopathy, macular edema, andproliferative retinopathy. As fluid leaks into the center of the macula,the part of the eye where sharp, straight-ahead vision occurs, thebuildup of fluid and the associated protein begin to deposit on or underthe macula. This deposit results in swelling that causes the subject'scentral vision gradually to become distorted. This condition is macularedema. Another condition that can occur is non-proliferative retinopathyin which vascular changes, such as microaneurysms, outside the macularregion of the eye can be observed.

These conditions can be associated with diabetic proliferativeretinopathy, which is characterized by increased neovascularization. Thenew blood vessels are fragile and are susceptible to bleeding. Theresult is scarring of the retina and occlusion or total blockage of thelight pathway through the eye due to the overformation of new bloodvessels. Subjects having diabetic macular edema often suffer from thenon-proliferative stage of diabetic retinopathy; however, subjects oftenonly begin to manifest macular edema at the onset of the proliferativestage.

Diabetic retinopathy is the most common cause of vision loss inworking-aged Americans. Severe vision loss occurs due to tractionalretinal detachments that complicate retinal neovascularization, but themost common cause of moderate vision loss is diabetic macular edema(DME).

VEGF is a hypoxia-regulated gene, and VEGF levels are increased inhypoxic or ischemic retina. Under most circumstances, Ang2 binds Tie2but does not stimulate phosphorylation, and acts as a Tie2 antagonist.In the eye, Ang2 is upregulated at sites of neovascularization and actsas a permissive factor for VEGF. Increased expression of VEGF in theretina does not stimulate sprouting of neovascularization from thesuperficial or intermediate capillary beds of the retina or thechoriocapillaris, but does stimulate sprouting from the deep capillarybed where constitutive expression of Ang2 occurs. Co-expression of VEGFand Ang2 at the surface of the retina causes sprouting ofneovascularization from the superficial retinal capillaries.

Angiogenesis, the process of creating new blood vessels frompre-existing vessels, is essential to a wide range of physiological andpathological events, including embryological development, menstruation,wound healing, and tumor growth. Most, if not all, tumors requireangiogenesis to grow and proliferate. VEGF can be a major factor inangiogenesis by increasing vessel permeability and capillary number.

VEGF is a protein that is primarily found in endothelial cells and hasfunctions in vasculogenesis, angiogenesis, and permeabilization of bloodvessels. The expression of VEGF is induced by hypoxia, activatedoncogenes, and cytokines. VEGF activation can lead to angiogenesis innormal human cells and tissues, but also angiogenesis in tumors andallows for tumor progression and growth. Inhibition of VEGF can inhibittumor growth leading to tumor regression. A variety of retinopathies areassociated with increased levels of VEGF; ischemia in the eye leads toan induction of VEGF production due to lack of oxygen. This increase inVEGF can cause hyperproliferation of blood vessels in the retina and canlead to blindness. The disclosed anti-VE-PTP (HPTP-β) antibodies can actto stabilize ocular vasculature and can counteract the stimulationcaused by VEGF and other inflammatory agents present in the diseasedretina. In some embodiments, administration of an anti-VE-PTP (HPTP-β)antibody to a subject can maintain the level of disease reversal afteradministration of anti-VEGF drugs to the subject has been withdrawn.

Macular degeneration is characterized by a gradual loss or impairment ofeyesight due to cell and tissue degeneration of the yellow macularregion in the center of the retina. Macular degeneration is oftencharacterized as one of two types, non-exudative (dry form) or exudative(wet form). Although both types are bilateral and progressive, each typecan reflect different pathological processes. The wet form ofage-related macular degeneration (AMD) is the most common form ofchoroidal neovascularization and a leading cause of blindness in theelderly. AMD affects millions of Americans over the age of 60, and isthe leading cause of new blindness among the elderly.

Choroidal neovascular membrane (CNVM) is a problem that is related to awide variety of retinal diseases, but is most commonly linked toage-related macular degeneration. With CNVM, abnormal blood vesselsstemming from the choroid (the blood vessel-rich tissue layer justbeneath the retina) grow up through the retinal layers. These newvessels are very fragile and break easily, and cause blood and fluid topool within the layers of the retina.

Diabetes (diabetes mellitus) is a metabolic disease caused by theinability of the pancreas to produce or use insulin. The most commontypes of diabetes are type 1 diabetes (often referred to as JuvenileOnset Diabetes Mellitus) and type 2 diabetes (often referred to as AdultOnset Diabetes Mellitus). Type 1 diabetes results from the body'sfailure to produce insulin due to loss of insulin producing cells, andpresently requires the person to inject insulin. Type 2 diabetesgenerally results from insulin resistance, a condition in which cellsfail to use insulin properly. Diabetes can be correlated to a largenumber of other conditions, including conditions or diseases of the eyeincluding diabetic retinopathy and diabetic macular edema (DME).

Diabetic retinopathy is a complication of diabetes that results fromdamage to the blood vessels of the light-sensitive tissue at the back ofthe eye (retina). At first, diabetic retinopathy can cause no symptomsor only mild vision problems. Eventually diabetic retinopathy can resultin blindness. Diabetic retinopathy can develop in anyone who has type 1diabetes or type 2 diabetes.

At the earliest stage of non-proliferative retinopathy, microaneurysmsoccur in the retina's tiny blood vessels. As the disease progresses,more of these blood vessels become damaged or blocked and these areas ofthe retina send signals into the regional tissue to grow new bloodvessels for nourishment. This stage is called proliferative retinopathy.The new blood vessels grow along the retina and along the surface of theclear, vitreous gel that fills the inside of the eye. The vessels havethin, fragile walls and without timely treatment, the new blood vesselscan leak blood, for example, whole blood or some constituents thereof,and can result in severe vision loss and even blindness. Also, fluid canleak into the center of the macula, the part of the eye where sharp,straight-ahead vision occurs. The fluid and the associated protein beginto deposit on or under the macula swell the subject's central visionbecomes distorted. This condition is called macular edema and can occurat any stage of diabetic retinopathy, but is more likely to occur as thedisease progresses.

Uveitis is a condition in which the uvea becomes inflamed. The eye ishollow on the inside with three different layers of tissue surrounding acentral cavity. The outermost is the sclera (white coat of the eye) andthe innermost is the retina. The middle layer between the sclera and theretina is called the uvea. The uvea contains many of the blood vesselsthat nourish the eye. Complications of uveitis include glaucoma,cataracts, and new blood vessel formation (neovascularization).

Ocular trauma is any sort of physical or chemical injury to the eye.Ocular trauma can affect anyone and major symptoms include redness orpain in the affected eye. The symptoms tend not to occur if tinyprojectiles cause the trauma.

Surgery-induced edema is the development of swelling in the eye tissuesfollowing surgery on the retina or other part of the eye. Cystoidmacular edema (CME) is an example of this phenomenon. CME can occur notonly in people who have had cataract surgery, but also those withdiabetes, retinitis pigmentosa, AMD, or conditions that cause chronicinflammation in the eye. The major symptoms of CME are blurred ordecreased central vision.

Ocular ischemic syndrome (OIS) encompasses the signs and symptoms thatresult from chronic vascular insufficiency. The condition is caused byocular hypoperfusion due to occlusion or stenosis of the common orinternal carotid arteries. OIS generally affects subjects of age 50-80,who often exhibit systemic diseases, such as hypertension or diabetes.The major symptoms of OIS are orbital pain, vision loss, changes of thevisual field, asymmetric cataract, and sluggish reaction to light.

Retinal vein occlusion (RVO) is the most common retinal vascular diseaseafter diabetic retinopathy. Depending on the area of retinal venousdrainage effectively occluded, the condition is broadly classified ascentral retinal vein occlusion (CRVO), hemispheric retinal veinocclusion (HRVO), or branch retinal vein occlusion (BRVO). Presentationof RVO is with variable painless visual loss with any combination offundal findings consisting of retinal vascular tortuosity, retinalhemorrhages (blot and flame shaped), cotton wool spots, optic discswelling and macular edema. In a CRVO, retinal hemorrhages can be foundin all four quadrants of the fundus, while the hemorrhages arerestricted to either the superior or inferior fundal hemisphere in aHRVO. In a BRVO, hemorrhages are largely localized to the area drainedby the occluded branch retinal vein. Vision loss occurs secondary tomacular edema or ischemia.

Compositions of the disclosure act to stabilize ocular vasculature and,in some embodiments, an agent of the disclosure can counteract thestimulation caused by VEGF and other inflammatory agents that can bepresent in the diseased retina. In some embodiments, administration ofan antibody of the disclosure to a subject can be used to maintain thelevel of disease reversal after administration of anti-VEGF drugs to thesubject has been withdrawn.

Treatment of Subjects

In some embodiments, a single administration of the composition of thedisclosure in a subject having a disease or condition as disclosedherein results in sustained intraocular expression of a VE-PTP (HPTP-β)inhibitor at a level sufficient for long-term suppression of ocularneovascularization.

For example, the level of VE-PTP (HPTP-β) inhibitor produced in a hostocular cell can be at least 100 pg/mL, at least 200 pg/mL, at least 300pg/mL, at least 400 pg/mL, at least 500 pg/mL, at least 600 pg/mL, atleast 00 pg/mL, at least 800 pg/mL, at least 900 pg/mL, at least 1000pg/mL, at least 2000 pg/mL, at least 3000 pg/mL, at least 4000 pg/mL, atleast 5000 pg/mL, at least 6000 pg/mL, at least 7000 pg/mL, at least8000 pg/mL, at least 9000 pg/mL or at least 10,000 pg/mL. The level ofVE-PTP (HPTP-β) inhibitor produced in host ocular cell can be at most100 pg/mL, at most 200 pg/mL, at most 300 pg/mL, at most 400 pg/mL, atmost 500 pg/mL, at most 600 pg/mL, at most 700 pg/mL, at most 800 pg/mL,at most 900 pg/mL, at most 1000 pg/mL, at most 2000 pg/mL, at most 3000pg/mL, at most 4000 pg/mL, at most 5000 pg/mL, at most 6000 pg/mL, atmost 7000 pg/mL, at most 8000 pg/mL, at most 9000 pg/mL, or at most10,000 pg/mL.

Protein levels can be measured at least about 0.1, at least about 0.2,at least about 0.3, at least about 0.4, at least about 0.5, at leastabout 0.6, at least about 0.7, at least about 0.8, at least about 0.9,at least about 1, at least about 2, at least about 3, at least about 4,at least about 5, at least about 6, at least about 7, at least about 14,at least about 21, at least about 30, at least about 50, at least about75, at least about 100, at least about 125, at least about 150, at leastabout 175, at least about 200, at least about 225, at least about 250,at least about 275, at least about 300, at least about 325, at leastabout 350, or at least about 365 days after administering apharmaceutical composition of the disclosure. Protein levels can bemeasured at most about 0.1, at most about 0.2, at most about 0.3, atmost about 0.4, at most about 0.5, at most about 0.6, at most about 0.7,at most about 0.8, at most about 0.9, at most about 1, at most about 2,at most about 3, at most about 4, at most about 5, at most about 6, atmost about 7, at most about 14, at most about 21, at most about 30, atmost about 50, at most about 75, at most about 100, at most about 125,at most about 150, at most about 175, at most about 200, at most about225, at most about 250, at most about 275, at most about 300, at mostabout 325, at most about 350, or at most about 365 days afteradministering a pharmaceutical composition of the disclosure.

Central Foveal Thickness

Also disclosed herein are methods for decreasing the Central FovealThickness (CFT) in a subject having a disease or condition as disclosedherein. The method comprises administering to an eye an antibodytargeting VE-PTP (HPTP-β), wherein the administration of the antibodycan be conducted in any manner.

The level of decrease in Central Foveal Thickness can be for example,from about 50 μm to about 1000 μm. The level of decrease in CentralFoveal Thickness can be for example, from about 50 μm to about 500 μm,from about 50 μm to about 750 μm, from about 150 μm to about 500 μm,from about 200 μm to about 500 μm, from about 200 μm to about 1000 μm,from about 250 μm to about 650 μm, or from about 400 μm to about 700 μm.

Visual Acuity

Further disclosed herein are methods for increasing the visual acuity ofa subject having a disease or condition as disclosed herein.

Visual acuity is acuteness or clearness of vision, which depends on thesharpness of the retinal focus within the eye and the sensitivity of theinterpretative faculty of the brain. Visual acuity is a measure of thespatial resolution of the visual processing system. Visual acuity istested by requiring the subject to identify characters, typicallynumbers or letters, on a chart from a set distance. Chart characters arerepresented as black symbols against a white background. The distancebetween the subject's eyes and the testing chart is set at a sufficientdistance to approximate infinity in the way the lens attempts to focus.Twenty feet, or six meters, is essentially infinity from an opticalperspective. In the present disclosure, an improvement in visual acuitywas assessed by an increase in the number of letters that can be readfrom the chart.

One non-limiting test for measuring visual acuity is the use of theESV-3000 ETDRS testing device and self-calibrated test lighting. TheESV-3000 device incorporates LED light source technology. Theauto-calibration circuitry constantly monitors the LED light source andcalibrates the test luminance to 85 cd/m² or 3 cd/m².

Although designed for clinical trials, where large-format ETDRS testing(up to 20/200) is performed at 4 meters, the device can be used in anon-research setting, i.e., hospital or clinic where ocular diseasemonitoring is conducted. To evaluate ETDRS properly, the test should beconducted under standardized lighting conditions, for example, photopictest level of 85 cd/m². Scoring of visual acuity can be accomplished inany manner chosen by the monitor. After providing a baseline evaluation,the increase or decrease in the number of letters that can be identifiedby the test subject provides a measure of sight increase or decreaseduring treatment.

Disclosed herein is a method for increasing visual acuity in a subjecthaving a disease or condition of the eye disclosed herein. This methodcomprises administering to a subject having the disease or condition ofthe eye, an antibody targeting VE-PTP (HPTP-β), wherein theadministration of the antibody can be conducted in any manner describedherein. The increase in the number of letters recognized by a treatedeye can be, for example, from about 1 to about 30 letters, from about 5to about 25 letters, from about 5 to about 20 letters, from about 5 toabout 15 letters, from about 5 to about 10 letters, from about 10 toabout 25 letters, from about 15 to about 25 letters, or from about 20 toabout 25 letters. The increase in visual acuity can be about 1 letter,about 5 letters, about 10 letters, about 15 letters, about 20 letters,or about 25 letters.

EXAMPLES Example 1

Generation of Monoclonal Antibodies to the VE-PTP (HPTP-β) ExtracellularDomain Using Hybridoma Technology

Hybridoma technology was used to generate monoclonal antibodies againstthe N-terminal extracellular domain (ECD) of human VE-PTP (HPTP-β) ormouse VE-PTP as shown in the schematic of FIG. 3. Mice were challengedwith human VE-PTP (HPTP-β) ECD protein to generate antibodies againstthe target protein. B-cells were then harvested from the spleen andlymph nodes and fused with myeloma cells to produce the hybridoma cellline. The fused cells were cultured in a medium where only myeloma cellsthat have genes derived from the antigen exposed mouse can grow.Colonies that were derived from a single myeloma cell were generated bylimiting dilution. Antibodies produced by the hybridoma colonies werethen screened for binding to human VE-PTP (HPTP-β) ECD by ELISA. Onehybridoma, R15E6, was selected for further analysis. R15E6 antibodybound to endogenous human VE-PTP (HPTP-β) from human umbilical veinendothelial cells (HUVECs) as shown by immunoprecipitation (IP) westernblot (left panel) and flow cytometry (right panel) in FIG. 4. No bindingof R15E6 to the bead control (protein A/G beads without antibody; BC) orVEGFR2 or Tie2 (R2/T2) was observed.

Example 2

Tie2 Activation in HUVECS with R15E6

R15E6 activated Tie2 in a concentration-dependent manner as demonstratedby IP experiments shown in panels A and B of FIG. 6. The antibody alsoenhanced the viability of serum starved endothelial cells in aconcentration-dependent manner in HUVECs as shown in panel C.

Example 3

Tie2 Activation and Cellular Permeability Experiments with MurineMonoclonal and Polyclonal Anti-Mouse VE-PTP ECD Antibodies

To characterize therapeutic effects of a VE-PTP antibody in vitro, Tie2activation and cellular permeability experiments were performed usinganti-mouse VE-PTP antibodies. Monoclonal and polyclonal antibodiesagainst mouse VE-PTP ECD were generated by immunizing rats with aVE-PTP-Fc fusion protein of the N-terminal 8 FN3 repeats of theextracellular domain. Immunization, hybridoma-fusion, and screening wereperformed as described above. In Example 3, monoclonal antibody 109.1(mAb 109.1) and polyclonal antibodies (pAbs) against the extracellularfibronectin type III (FN3)-like domains 1-8 of mouse VE-PTP wereselected for further analysis.

FIG. 7 illustrates the effect of mAb 109.1 on Tie2 activation (top leftpanel), mouse VE-PTP protein expression (bottom left panel), and Akt(right panel) in mouse endothelial cells (bEnd) as determined by westernblot. Treatment with mAb 109.1 resulted in the activation of Tie2 asindicated by Tie2 phosphorylation (pTyr; top left panel). Akt wasconstitutively activated in the tested mouse endothelial cells asconfirmed by the absence of increased phosphorylation with increasingconcentrations of antibody (right panel).

In vitro experiments with mouse pAb 1-8 also enhanced Tie2 (Tie-2)activation and downstream signaling in endothelial cells. Cultured mouseendothelial (bEnd.5) cells were treated with pAb 1-8 or preimmune(control) antibodies for 1 hr and subsequently immunoprecipitated forTie2, followed by immunoblotting with anti-phosphotyrosine antibodies(pTyr) and antibodies against Tie2. Aliquots of cell lysates withidentical protein content were directly immunoblotted for VE-PTP andTie2 (bottom panels). FIG. 8 illustrates the immunoprecipitationexperiments in which pAbs rapidly induced Tie2 activation in bEnd.5cells treated with polyclonal antibodies against mouse VE-PTP orpreimmune antibodies for 1 h (panel A) or 3 min (panel B).

Consistent with the vascular-stabilizing effects of Tie2 activation,VE-PTP ECD antibodies reduced thrombin- and VEGF-induced permeability ofendothelial monolayers in vitro as shown in FIG. 9. Paracellularpermeability for 250 kD FITC-dextran was determined for cultured HUVECmonolayers grown in transwell filters. Permeability was induced eitherwith thrombin (panel A) or VEGF (panel B). Permeability of PBS-treatedcells was set to 100%. For VE-PTP targeting, cells were treated withanti-VE-PTP pAbs. As a control, cells were treated with preimmuneantibodies (control Ab). The data represent two independent experiments.

Consistent with the vascular-stabilizing effects of Tie2 activation,VE-PTP ECD antibodies also blocked VEGF-induced cutaneous vascularpermeability in vivo as shown in FIG. 10. Both monoclonal and polyclonalVE-PTP ECD antibodies, mAb 109.1 (panel A) and pAb PTP 1-8 (panel B),inhibited VEGF-induced cutaneous vascular permeability as demonstratedby the Miles assay. The Miles assay can be used as an in vivo model ofvascular permeability, which is characteristic of vascular leakage andneovascularization. Mice were injected intravenously with 100 μg ofcontrol IgG or anti-mouse VE-PTP antibodies 30 min before starting theMiles assay. Evan's blue was then injected to the mice intravenously,followed by intradermal injection of VEGF (striped bars) or PBS (solidblack bars) after 10 min. After 30 min, the mice were sacrificed and thedye was extracted from the skin samples and quantified. Data sets werechecked for normality and equal variance. Values are presented asmeans±standard error of mean (SEM). As shown in panel C, the blockade ofpermeability correlates with Tie2 phosphorylation as demonstrated inlung tissue of mice treated with pAb PTP 1-8.

Example 4

Effects of a Mouse VE-PTP ECD Monoclonal Antibody on Retinal andChoroidal Neovascularization in Mice

In an ischemic retinopathy model that mimics the aspects ofproliferative diabetic retinopathy, a VE-PTP ECD monoclonal antibody(mAb 109.1) significantly reduced retinal neovascularization afteradministration of the antibody as shown in FIG. 11 (panel A) andquantified in FIG. 12 (panel A). At P12, mice with ischemic retinopathywere administered an intravitreous injection of 0.1, 0.5, or 2 μg of mAb109.1 or 2 μg IgG isotype control (n≥12 for each). At P17, extensiveGSA-stained retinal neovascularization was observed in controlIgG-injected eyes and significantly less retinal neovascularization wasobserved in eyes injected with 2 μg anti-VE-PTP. *P<0.001.

Similarly, in two models that mimic different aspects of wet age-relatedmacular edema (retinal angiomatous proliferation and choroidalneovascularization), a single 2 μg intravitreal administration of theVE-PTP ECD monoclonal antibody (mAb 109.1) significantly reduced bothretinal neovascularization (panel B of FIG. 11 and quantified in panel Bof FIGURE 12). At P15, six Rho-VEGF transgenic mice were given anintravitreous injection of 0.5 or 2 μg of mAb 109.1 in one eye and acorresponding dose of control IgG in the fellow eye. At P21,significantly less GSA-stained subretinal neovascularization wasobserved in the eyes treated with 0.5 or 2 μg of mAb 109.1 than in thecontrol IgG-treated eyes. *P=0.01 by unpaired t-test for comparison withIgG control fellow eyes. Scale bar: 100 μm.

Intravitreous injection of 2 μg of mAb 109.1 significantly reduced thearea of choroidal neovascularization at Bruch's membrane rupture sitescompared with control IgG (panel C of FIG. 11 and quantified in panel Cof FIG. 12). *P<0.001 by 1-way ANOVA with Bonferroni's correction. Scalebar: 100 μm.

Subcutaneous administration of mAb 109.1 also reduced ischemicneovascularization in an ischemic retinopathy mouse model. The mean areaof retinal neovascularization was reduced by about 30% compared tovehicle, as shown in FIG. 25. Mice with ischemic retinopathy wereadministered a subcutaneous injection of 2 mg/kg q.o.d. (every otherday)×3.

Example 5

Effects of a Mouse VE-PTP ECD Monoclonal Antibody in Combination with anAnti-VEGF Agent on Retinal Detachment in Mice

To evaluate the effects of combination therapies using an anti-VE-PTPantibody disclosed herein, a Tet/opsin/VEGF mouse model was used.Tet/opsin/VEGF transgenic mice develop severe subretinalneovascularization and vascular leakage, and leads to exudative retinaldetachment mediated by inducible retinal overexpression of human VEGF.FIG. 24 shows enhanced efficacy of the combination of an anti-mouseVE-PTP antibody (mAb 109.1) and aflibercept compared to either therapyalone to prevent retinal detachment in the mice, as denoted by thedouble asterisks. Rat IgG was used as the control.

Example 6

Generation and Purification of Humanized Antibodies from R15E6 AgainstHuman VE-PTP (HPTP-β)

R15E6 is a 150 kDa antibody containing heavy chains and light chains.Sixteen different humanized variations of the antibody were synthesizedusing different combinations of heavy chain sequence to light chainsequences as shown in TABLE 12.

R15E6 humanized variants, as described in Example 1, were cloned intothe pVitro DHFR3 mammalian expression vector. Medium-scale transienttransfection expression analysis was carried out to determine expressionyield from CHO cells. Suspension-adapted CHO cells were cultivated at2.0-3.0×10⁵ cells/mL with 85% CO₂ at 37° C. at 150 rpm in Pro CHO4serum-free medium supplemented with 8 mM L-glutamine and 10 mL/Lhypoxanthine/thymidine in 500 mL vented flasks. Maxi-preps of eachconstruct were prepared using a Nucleobond pc500 kit according tomanufacturer instructions. Vector DNA was quantified using a NanoDropLite™ spectrophotometer.

100 mL of cells at a final density of 1.0×10⁶ cells/mL were transientlytransfected with 1.25 μg/mL of plasmid DNA in Pro CHO5 serum-free mediumsupplemented with 8 mM L-glutamine and 10 mL/L hypoxanthine/thymidine in500 mL vented flasks. Transfected cultures were incubated for 8 to 11days with 85% CO₂ at 37° C. at 150 rpm before harvesting bycentrifugation at 4,000 rpm at 4° C. for 40 min.

Following centrifugation, the media were filtered through a 0.8 μmcellulose acetate filter. Each batch was purified using an AmershamBiosciences AKTA chromatography system. Expressed humanized antibodieswere purified via Protein A affinity chromatography. A 1-mL HiTrapProtein A column was used for all humanized antibody purifications. Allpurifications were carried out using Fusion Antibodies in-house wash andelution buffers. After loading of the protein into the column, any boundantibody was eluted using a glycine/Tris buffer (pH 3). All eluted 1 mLfractions were neutralized with 100 μL Tris buffer (pH 8.5). Elutedfractions corresponding to the elution peak were selected for overnightdialysis in PBS. Following dialysis, the concentration of the antibodywas measured with a NanoDrop Lite™ spectrophotometer.

Example 7

Evaluation of Humanized Antibodies Against Human VE-PTP (HPTP-β)

Samples of the humanized antibodies were analyzed by reducing andnon-reducing SDS-PAGE (FIG. 13) to determine the presence of heavy andlight chains and the purity of the antibodies. The mouse/human chimericantibody, HC0LC0, was derived from R15E6 by fusion of the murine heavyand light chain variable regions to the human Fc region. In bothSDS-PAGE gels, lane 1: SeeBlue Plus2 pre-stained protein standard; lane2: HC0LC0 #1; lane 3: HC1LC1 #2; lane 4: HC1LC2 #1; lane 5: HC1LC3 #1;lane 6: HC1LC3 #2; lane 7: HC1LC4 #1; lane 8: HC2LC1 #1; lane 9: HC2LC2#1; lane 10: HC2LC3 #1; lane 11: HC2LC3 #2; lane 12: HC2LC4 #1; lane 13:HC3LC1 #1; lane 14: HC3LC2 #1; lane 15: HC3LC3 #1; lane 16: HC3LC4 #1;lane 17: HC4LC1 #1; lane 18: HC4LC1 #2; lane 19: HC4LC2 #1; lane 20:HC4LC3 #1; lane 21: HC4LC3 #2; lane 22: HC4LC4 #1; lane 23: HC4LC4 #2;and lane 24: SeeBlue Plus2 pre-stained protein standard. The SDS-PAGEgel analysis showed high purity of all analyzed samples. In the reducingSDS-PAGE, the heavy and light chains are visible and are at the expectedmolecular weight of 50 kDa for the heavy chain and 25 kDa for the lightchain. In the non-reducing SDS-PAGE, no free heavy or light chains wereobserved at around 50 kDa or 25 kDa, respectively. The gels show a lowamount of protein for all HC4 variants and HC1LC3, HC2LC3, and HC3LC3variants.

Purified R15E6 was quantified using the extinction coefficient of 13.7as the standard reference for IgG at 280 nm using a NanoDrop Lite™spectrophotometer. TABLE 16 summarizes the results of the SDS-PAGEanalysis. In agreement with the SDS-PAGE analysis, all of the heavychain 4 variants expressed little to no antibody.

TABLE 16 Concentration Total yield R15E6 Variant Volume (mg/ml) (mg) SDSPAGE HC0 LC0 #1 100 ml 0.403 0.85 HC and LC present HC1 LC1 #1 100 ml 00 No antibody purified HC1 LC1 #2 160 ml 0.141 0.28 HC and LC presentHC1 LC2 #1 100 ml 0.127 0.23 HC and LC present HC1 LC3 #1 100 ml 0.0450.086 HC and LC present faint HC1 LC3 #2 160 ml 0.175 0.39 HC and LCpresent HC1 LC4 #1 100 ml 0.1 0.18 HC and LC present HC2 LC1 #1 100 ml0.147 0.28 HC and LC present HC2 LC2 #1 100 ml 0.538 1.08 HC and LCpresent HC2 LC3 #1 100 ml 0.034 0.068 HC and LC present faint HC2 LC3 #2160 ml 0.591 1.18 HC and LC present HC2 LC4 #1 100 ml 0.165 0.39 HC andLC present HC3 LC1 #1 100 ml 0.078 0.16 HC and LC present HC3 LC2 #1 100ml 0.264 0.53 HC and LC present HC3 LC3 #1 100 ml 0.043 0.07 HC and LCvery faint HC3 LC4 #1 100 ml 0.406 0.812 HC and LC present HC4 LC1 #1100 ml 0.025 0.06 No bands HC4 LC1 #2 160 ml 0.051 0.11 No bands HC4 LC2#1 100 ml 0.051 0.11 No bands HC4 LC3 #1 100 ml 0.053 0.13 No bands HC4LC3 #2 160 ml 0.035 0.07 No bands HC4 LC4 #1 100 ml 0.041 0.09 No bandsHC4 LC4 #2 160 ml 0.031 0.068 No bands

Example 8

Selectivity of R15E6 and Humanized Variants Against Human VE-PTP(HPTP-β), Cynomologus VE-PTP (Cyno PTP-β), and Human HPTP-η

Tissue culture supernatants containing the antibodies from hybridomaswere tested against targets human VE-PTP (Human Beta ½ ECD), cyno VE-PTP(Cyno Beta), and human HPTP-η (Human Eta) to determine binding affinityby ELISA.

Human HPTP-β, cyno PTPβ, and human HPTP-η were independently producedand purified using HEK293 cells. The HEK293 cells were seeded in ashaker flask for 24 hrs before transfection with plasmids expressingspecies-specific VE-PTP or HPTP-η. The HEK293 cells were grown usingserum-free chemically defined media. The DNA for the species-specificVE-PTP or HPTP-η construct was transiently transfected into a 30 mLsuspension of HEK293. After 24 hrs, the cells were counted to assess theviability of the cells and determine the viable cell count. Additionalreadings were taken throughout the transient transfection process. Thecell culture was harvested at day 5 of the transient transfection. Theconditioned media supernatant harvested from the transient transfectionwas clarified by centrifugation. The supernatant was filtered using a0.2 μm membrane filter. The protein was then purified by anti-His tagaffinity chromatography. After purification, buffer exchange of theprotein to PBS (pH 7.4) was performed. Then, SDS-Gel CapillaryElectrophoresis (CE-SDS) analysis was performed and the protein waspurified. The amino acid sequences of the DNA plasmid inserts, the DNAplasmid inserts, and corresponding wildtype sequences are shown in TABLE15 below. The signal peptide of the protein sequences is underlined.

TABLE 15 SEQ ID NO: Name Sequence 38 ProteinMEWSWVFLFFLSVTTGVHSEPERCNFTLAESKASSHSVS sequence ofIQWRILGSPCNFSLIYSSDTLGAALCPTFRIDNTTYGCN Human VE-PTPLQDLQAGTIYNFKIISLDEERTVVLQTDPLPPARFGVSK (HPTP-β)EKTTSTGLHVWWTPSSGKVTSYEVQLFDENNQKIQGVQI containing firstQESTSWNEYTFFNLTAGSKYNIAITAVSGGKRSFSVYTN eight FN3GSTVPSPVKDIGISTKANSLLISWSHGSGNVERYRLMLM repeats withDKGILVHGGVVDKHATSYAFHGLTPGYLYNLTVMTEAAG C-terminalLQNYRWKLVRTAPMEVSNLKVTNDGSLTSLKVKWQRPPG His₆ tagNVDSYNITLSHKGTIKESRVLAPWITETHFKELVPGRLY (SEQ ID NO: 52)QVTVSCVSGELSAQKMAVGRTFPLAVLQLRVKHANETSL (Human ½ VE-SIMWQTPVAEWEKYIISLADRDLLLIHKSLSKDAKEFTF PTP ECD)TDLVPGRKYMATVTSISGDLKNSSSVKGRTVPAQVTDLHVANQGMTSSLFTNWTQAQGDVEFYQVLLIHENVVIKNESISSETSRYSFHSLKSGSLYSVVVTTVSGGISSRQVVVEGRTVPSSVSGVTVNNSGRNDYLSVSWLLAPGDVDNYEVTLSHDGKVVQSLVIAKSVRECSFSSLTPGRLYTVTITTRSGKYENHSFSQERTVPDKVQGVSVSNSARSDYLRVSWVHATGDFDHYEVTIKNKNNFIQTKSIPKSENECVFVQLVPGRLYSVTVTTKSGQYEANEQGNGRTIPEKGNSADIQHSGGRS SLEGPRFERTGGGHHHHHH 39 Wild-typeMLSHGAGLALWITLSLLQTGLAEPERCNFTLAESKASSH proteinSVSIQWRILGSPCNFSLIYSSDTLGAALCPTFRIDNTTY sequence ofGCNLQDLQAGTIYNFRIISLDEERTVVLQTDPLPPARFG Human VE-PTPVSKEKTTSTSLHVWWTPSSGKVTSYEVQLFDENNQKIQG (HPTP-β)VQIQESTSWNEYTFFNLTAGSKYNIAITAVSGGKRSFSVYTNGSTVPSPVKDIGISTKANSLLISWSHGSGNVERYRLMLMDKGILVHGGVVDKHATSYAFHGLTPGYLYNLTVMTEAAGLQNYRWKLVRTAPMEVSNLKVTNDGSLTSLKVKWQRPPGNVDSYNITLSHKGTIKESRVLAPWITETHFKELVPGRLYQVTVSCVSGELSAQKMAVGRTFPDKVANLEANNNGRMRSLVVSWSPPAGDWEQYRILLFNDSVVLLNITVGKEETQYVMDDTGLVPGRQYEVEVIVESGNLKNSERCQGRTVPLAVLQLRVKHANETSLSIMWQTPVAEWEKYIISLADRDLLLIHKSLSKDAKEFTFTDLVPGRKYMATVTSISGDLKNSSSVKGRTVPAQVTDLHVANQGMTSSLFTNWTQAQGDVEFYQVLLIHENVVIKNESISSETSRYSFHSLKSGSLYSVVVTTVSGGISSRQVVVEGRTVPSSVSGVTVNNSGRNDYLSVSWLLAPGDVDNYEVTLSHDGKVVQSLVIAKSVRECSFSSLTPGRLYTVTITTRSGKYENHSFSQERTVPDKVQGVSVSNSARSDYLRVSWVHATGDFDHYEVTIKNKNNFIQTKSIPKSENECVFVQLVPGRLYSVTVTTKSGQYEANEQGNGRTIPEPVKDLTLRNRSTEDLHVTWSGANGDVDQYEIQLLENDMKVEPPFHLVNTATEYRFTSLTPGRQYKILVLTISGDVQQSAFIEGFTVPSAVKNIHISPNGATDSLTVNWTPGGGDVDSYTVSAFRHSQKVDSQTIPKHVFEHTFHRLEAGEQYQEVIIASVSGSLKNQINVVGRTVPASVQGVIADNAYSSYSLIVSWQKAAGVAERYDILLLTENGILLRNTSEPATTKQHKFEDLTPGKKYKIQILTVSGGLFSKEAQTEGRTVPAAVTDLRITENSTRHLSFRWTASEGELSWYNIFLYNPDGNLQERAQVDPLVQSFSFQNLLQGRMYKMVIVTHSGELSNESFIFGRTVPASVSHLRGSNRNTTDSLWFNWSPASGDFDFYELILYNPNGTKKENWKDKDLTEWRFQGLVPGRKYVLWVVTHSGDLSNKVTAESRTAPSPPSLMSFADIANTSLAITWKGPPDWTDYNDFELQWLPRDALTVFNPYNNRKSEGRIVYGLRPGRSYQFNVKTVSGDSWKTYSKPIFGSVRTKPDKIQNLHCRPQNSTAIACSWIPPDSDFDGYSIECRKMDTQEVEFSRKLEKEKSLLNIMMLVPHKRYLVSIKVQSAGMTSEVVEDSTITMIDRPPPPPPHIRVNEKDVLISKSSINETVNCSWFSDTNGAVKYFTVVVREADGSDELKPEQQHPLPSYLEYRHNASIRVYQTNYFASKCAENPNSNSKSFNIKLGAEMESLGGKCDPTQQKFCDGPLKPHTAYRISIRAFTQLFDEDLKEFTKPLYSDTFFSLPITTESEPLFGAIEGVSAGLFLIGMLVAVVALLICRQKVSHGRERPSARLSIRRDRPLSVHLNLGQKGNRKTSCPIKINQFEGHFMKLQADSNYLLSKEYEELKDVGRNQSCDIALLPENRGKNRYNNILPYDATRVKLSNVDDDPCSDYINASYIPGNNFRREYIVTQGPLPGTKDDFWKMVWEQNVHNIVMVTQCVEKGRVKCDHYWPADQDSLYYGDLILQMLSESVLPEWTIREFKICGEEQLDAHRLIRHFHYTVWPDHGVPETTQSLIQFVRTVRDYINRSPGAGPTVVHCSAGVGRTGTFIALDRILQQLDSKDSVDIYGAVHDLRLHRVHMVQTECQYVYLHQCVRDVLRARKLRSEQENPLFPIYENVNPEY HRDPVYSRH 40 DNA sequenceATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTA of Human ½ACGACTGGTGTCCACTCCGAGCCCGAGAGATGCAACTTC VE-PTP ECDACCCTGGCCGAGTCCAAGGCCTCCTCCCACTCCGTGTCT containingATCCAGTGGCGGATCCTGGGCTCCCCCTGCAACTTCTCT first eightCTGATCTACTCCTCCGACACCCTGGGCGCTGCCCTGTGC FN3 repeatsCCTACCTTCAGAATCGACAACACCACCTACGGCTGCAAC (SEQ ID NO: 38)CTGCAGGATCTGCAGGCCGGCACCATCTACAACTTCAAGATCATCTCCCTGGACGAGGAACGGACCGTGGTGCTGCAGACCGATCCTCTGCCTCCTGCCAGATTCGGCGTGTCCAAAGAAAAGACCACCTCCACCGGACTGCACGTGTGGTGGACCCCTTCCAGCGGCAAAGTGACCTCCTACGAGGTGCAGCTGTTCGACGAGAACAACCAGAAAATCCAGGGCGTGCAGATCCAGGAATCCACCTCCTGGAACGAGTACACCTTCTTCAACCTGACCGCCGGCTCCAAGTACAATATCGCCATCACCGCCGTGTCCGGCGGCAAGAGATCCTTCTCCGTGTACACCAACGGCTCCACCGTGCCCAGCCCCGTGAAGGACATCGGCATCTCCACCAAGGCCAACTCCCTGCTGATCTCCTGGTCCCACGGCTCCGGCAACGTGGAACGGTACAGACTGATGCTGATGGACAAGGGCATCCTGGTGCACGGCGGCGTGGTGGATAAGCACGCCACCTCTTACGCCTTCCACGGCCTGACCCCTGGCTACCTGTACAATCTGACCGTGATGACCGAGGCCGCTGGACTGCAGAACTACCGGTGGAAGCTCGTGCGGACCGCCCCCATGGAAGTGTCCAACCTGAAAGTGACCAACGACGGCTCCCTGACCTCTCTGAAAGTGAAGTGGCAGAGGCCCCCTGGCAATGTGGACTCCTACAACATCACCCTGTCCCACAAGGGCACCATCAAAGAATCCCGGGTGCTGGCCCCTTGGATCACCGAGACACACTTCAAAGAACTGGTGCCTGGCCGGCTGTACCAAGTGACCGTGTCCTGTGTGTCTGGCGAGCTGTCCGCCCAGAAAATGGCCGTGGGCAGAACCTTCCCTCTGGCCGTGCTGCAGCTGAGAGTGAAGCACGCTAACGAGACATCCCTGTCCATCATGTGGCAGACCCCCGTGGCCGAGTGGGAGAAGTACATCATCAGCCTGGCCGACCGGGACCTGCTGCTGATCCACAAGTCCCTGAGCAAGGACGCCAAAGAGTTCACCTTCACCGACCTGGTGCCCGGCAGAAAGTACATGGCCACCGTGACCTCCATCTCCGGCGACCTGAAGAACTCCTCCAGCGTGAAGGGCAGGACCGTGCCTGCCCAAGTGACAGACCTGCACGTGGCCAACCAGGGCATGACCTCCTCCCTGTTCACCAACTGGACCCAGGCTCAGGGCGACGTGGAATTCTACCAGGTGCTGCTGATTCATGAGAACGTCGTGATCAAGAACGAGTCCATCTCCTCCGAGACAAGCCGGTACTCCTTCCACTCCCTGAAGTCCGGCAGCCTGTACTCCGTGGTCGTGACCACAGTGTCCGGGGGCATCTCCTCTAGACAGGTGGTGGTGGAAGGCCGCACCGTGCCTAGTTCAGTGTCAGGCGTGACCGTGAACAACAGCGGCCGGAACGACTACCTGTCCGTGTCTTGGCTGCTGGCTCCTGGGGACGTGGACAACTACGAAGTGACCCTGAGCCACGACGGCAAGGTGGTGCAGTCTCTCGTGATCGCCAAGTCCGTGCGCGAGTGCTCCTTCAGCTCTCTGACACCTGGCAGACTGTATACCGTGACCATCACCACCAGATCCGGGAAGTACGAGAACCACAGCTTCTCCCAGGAACGCACAGTGCCCGACAAGGTGCAGGGCGTGTCAGTGTCTAACTCCGCCAGATCTGACTACCTGCGGGTGTCCTGGGTGCACGCTACCGGCGACTTCGACCATTATGAAGTGACAATCAAGAACAAGAACAACTTCATCCAGACCAAGTCCATCCCCAAGTCCGAGAACGAGTGCGTGTTCGTGCAGCTGGTGCCAGGCAGACTGTACTCTGTGACAGTGACCACCAAGTCCGGCCAGTACGAGGCCAACGAGCAGGGCAACGGCAGGACCATCCCTGAGAAGGGCAACTCCGCCGACATCCAGCACTCTGGCGGCAGATCCTCTCTGGAAGGCCCCAGATTCGAGAGAACCGGCGGAGGC CACCACCATCATCACCATTGA 41 ProteinMEWSWVFLFFLSVTTGVHSERCNFTLAESKASSHSVSIR sequence ofWRIWGSPCNFNLTYSSDTLGAASCPPFRLDNTTYGCNLQ CynomolgusDLQAGTIYNFRIVSLDGEERTVVLQTDPLPPARFGVSKE VE-PTPKTTSTSLHVWWTPSPGKVTSYEVQLFDENNQKIQGVQIQ (Cyno PTP-β)ESTSWNKYTFFNLTAGSKYNITITAVSGGKRSSSVYTNG containingSTVPSPVKDIGISTKANSLLVSWSHGSGNVERYRLMLMD first eightKGILVHGSVVDRQATSYTFNGLTPGYLYNLTVVTEAAGL FN3 repeatsQNYKWKLVRTAPMEVSNLKVTNDGSLTSLKVKWQRPPGN with C-terminalVDSYNITLSHKGTIKESRVLAPRVTETHFKELTPGRLYQ His₁₀ tagVTVSCVSGELSAQRMAVGRTFPLPVLQLRVKHANETSLS (SEQ ID NO: 53)IIWQPPVAEWEEYIISLADRDLRLIHKSLSKDAKEFTFT (Cyno Beta ½DLVPGRKYMATVTSISGDLKNSSSVKGRTVPAQVTDLHV VE-PTP ECD)ANQGMTSSLFTNWTQAQGDVEFYQVLLIHENVVIKNESIPSETSGYNFHFLKSGSLYSVVVTTVSGGISSRQVVVEGRTVPSSVSGVTVNNSGRNDYLSVSWLPAPGDVDNYEVTLSHDGRVVQSLVIAKSVRECSFSSLTPGRLYTVTITTRSGKYENHSFSQERTVPDKVQGVSVSNSARSDYLRVSWVHATGDFDHYEVTIKNKNNFIETKSIPKSENECVFVQLVPGRLYSVTVTTKSGQYEASEQGNGRTGGGHHHHHHHHHH 42 DNA sequenceATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTA of Cyno Beta ½ACGACTGGTGTCCACTCCGAGCGGTGCAACTTTACCCTG VE-PTP ECDGCCGAGTCCAAGGCCTCCTCCCACTCCGTGTCTATCCGG containingTGGCGGATCTGGGGCTCCCCCTGCAACTTCAACCTGACC first eightTACTCCTCCGATACCCTGGGCGCTGCCTCCTGTCCTCCT FN3 repeatsTTCCGGCTGGACAACACCACCTACGGCTGCAACCTGCAG (SEQ ID NO: 41)GATCTGCAGGCCGGCACCATCTACAACTTCCGGATCGTGTCCCTGGACGGCGAGGAACGGACAGTGGTGCTGCAGACCGATCCTCTGCCCCCTGCCAGATTCGGCGTGTCCAAAGAAAAGACCACCTCCACCTCCCTGCACGTGTGGTGGACCCCTAGCCCTGGCAAAGTGACCTCCTACGAGGTGCAGCTGTTCGACGAGAACAACCAGAAAATCCAGGGCGTGCAGATCCAGGAATCCACCTCCTGGAACAAGTACACCTTCTTCAATCTGACCGCCGGCTCCAAGTACAACATCACCATCACCGCCGTGTCCGGCGGCAAGAGATCCTCCTCCGTGTACACCAACGGCTCCACCGTGCCCAGCCCCGTGAAGGACATCGGCATCTCCACCAAGGCCAACTCCCTGCTGGTGTCCTGGTCCCACGGCTCCGGCAACGTGGAACGGTACAGACTGATGCTGATGGACAAGGGCATCCTGGTGCACGGCAGCGTGGTGGATAGACAGGCCACCTCCTACACCTTCAACGGCCTGACCCCCGGCTACCTGTATAACCTGACCGTCGTGACCGAGGCCGCTGGACTGCAGAACTACAAGTGGAAGCTCGTGCGGACCGCCCCCATGGAAGTGTCCAACCTGAAAGTGACCAACGACGGCTCCCTGACCTCTCTGAAAGTGAAGTGGCAGAGGCCCCCTGGCAATGTGGACAGCTACAATATCACCCTGTCCCACAAGGGCACCATCAAAGAATCCCGGGTGCTGGCCCCCAGAGTGACCGAGACACACTTCAAAGAGCTGACCCCTGGCCGGCTGTACCAAGTGACCGTGTCCTGTGTGTCTGGCGAGCTGTCTGCCCAGAGAATGGCCGTGGGCAGAACCTTCCCTCTGCCCGTGCTGCAGCTGAGAGTGAAGCACGCCAACGAGACATCCCTGTCCATCATCTGGCAGCCCCCTGTGGCCGAGTGGGAAGAGTACATCATCAGCCTGGCCGACCGGGACCTGCGGCTGATCCACAAGTCCCTGAGCAAGGACGCCAAAGAGTTCACCTTCACCGACCTGGTGCCTGGCCGGAAGTACATGGCCACCGTGACCTCCATCTCCGGCGACCTGAAGAACTCCTCCAGCGTGAAGGGCAGGACCGTGCCTGCCCAAGTGACAGACCTGCATGTGGCCAACCAGGGCATGACCTCCAGCCTGTTCACCAACTGGACCCAGGCTCAGGGCGACGTGGAATTCTACCAGGTGCTGCTGATCCATGAGAACGTCGTGATCAAGAACGAGTCCATCCCCTCCGAGACAAGCGGCTACAACTTTCACTTCCTGAAGTCCGGCAGCCTGTACTCCGTGGTCGTGACCACAGTGTCCGGGGGCATCTCCTCTAGAAGGTGGTGGTGGAAGGCCGCACCGTGCCTAGTTCAGTGTCAGGCGTGACCGTGAACAACAGCGGCCGGAACGACTACCTGTCCGTGTCTTGGCTGCCTGCCCCTGGGGACGTGGACAACTACGAAGTGACCCTGTCTCACGACGGCCGGGTGGTGCAGTCTCTCGTGATCGCTAAGTCCGTGCGCGAGTGCTCCTTCAGCAGCCTGACACCTGGCAGACTGTATACCGTGACCATCACCACCAGATCCGGGAAGTACGAGAACCACAGCTTCTCCCAGGAACGAACCGTGCCCGACAAGGTGCAGGGCGTGTCAGTGTCTAACTCCGCCAGATCTGACTACCTGAGAGTGTCCTGGGTGCACGCCACCGGCGACTTCGACCATTATGAAGTGACAATCAAGAACAAGAACAACTTCATCGAGACAAAGAGCATCCCCAAGTCCGAGAACGAGTGCGTGTTCGTGCAGCTGGTGCCAGGCAGGCTGTATTCTGTGACAGTGACCACCAAGTCCGGCCAGTACGAGGCCTCTGAGCAGGGCAATGGCAGAACCGGCGGTGGACACCACC ATCATCACCATCACCACCATCACTAG 43Protein MEWSWVFLFFLSVTTGVHSAGGTPSPIPDPSVATVATGE sequence ofNGITQISSTAESFHKQNGTGTPQVETNTSEDGESSGAND Human HPTP-ηSLRTPEQGSNGTDGASQKTPSSTGPSPVFDIKAVSISPT with C-terminalNVILTWKSNDTAASEYKYVVKHKMENEKTITVVHQPWCN His₁₀ tagITGLRPATSYVFSITPGIGNETWGDPRVIKVITEPIPVS (SEQ ID NO: 53)DLRVALTGVRKAALSWSNGNGTASCRVLLESIGSHEELT (Human HPTP-ηQDSRLQVNISGLKPGVQYNINPYLLQSNKTKGDPLGTEG ECD)GLDASNTERSRAGSPTAPVHDESLVGPVDPSSGQQSRDTEVLLVGLEPGTRYNATVYSQAANGTEGQPQAIEFRTNAIQVFDVTAVNISATSLTLIWKVSDNESSSNYTYKIHVAGETDSSNLNVSEPRAVIPGLRSSTFYNITVCPVLGDIEGTPGFLQVHTPPVPVSDFRVTVVSTTEIGLAWSSHDAESFQMHITQEGAGNSRVEITTNQSIIIGGLFPGTKYCFEIVPKGPNGTEGASRTVCNRTVPSAVFDIHVVYVTTTEMWLDWKSPDGASEYVYHLVIESKHGSNHTSTYDKAITLQGLIPGTLYNITISPEVDHVWGDPNSTAQYTRPSNVSNIDVSTNTTAATLSWQNFDDASPTYSYCLLIEKAGNSSNATQVVTDIGITDATVTELIPGSSYTVEIFAQVGDGIKSLEPGRKSFCTDPASMASFDCEVVPKEPALVLKWTCPPGANAGFELEVSSGAWNNATHLESCSSENGTEYRTEVTYLNFSTSYNISITTVSCGKMAAPTRNTCTTGITDPPPPDGSPNITSVSHNSVKVKFSGFEASHGPIKAYAVILTTGEAGHPSADVLKYTYEDFKKGASDTYVTYLIRTEEKGRSQSLSEVLKYEIDVGNESTTLGYYNGKLEPLGSYRACVAGFTNITFHPQNKGLIDGAESYVSFSRYSDAVSLPQDPGVICGGGHHHHHHHHHH 44 DNA sequenceATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTA of HumanACGACTGGTGTCCACTCCGCAGGTGGCACCCCTAGTCCA HPTP-η withATTCCTGACCCTTCAGTAGCAACTGTTGCCACAGGGGAA C-terminalAATGGCATAACGCAGATCAGCAGTACAGCAGAATCCTTT His₁₀ tagCATAAACAGAATGGAACTGGAACACCTCAGGTGGAAACA (SEQ ID NO: 43)AACACCAGTGAGGATGGTGAAAGCTCTGGAGCCAACGAT (“His₁₀”AGTTTAAGAACACCTGAACAAGGATCTAATGGGACTGAT disclosed asGGGGCATCTCAAAAAACTCCCAGTAGCACTGGGCCCAGT SEQ ID NO: 53)CCTGTGTTTGACATTAAAGCTGTTTCCATCAGTCCAACCAATGTGATCTTAACTTGGAAAAGTAATGACACAGCTGCTTCTGAGTACAAGTATGTAGTAAAGCATAAGATGGAAAATGAGAAGACAATTACTGTTGTGCATCAACCATGGTGTAACATCACAGGCTTACGTCCAGCGACTTCATATGTATTCTCCATCACTCCAGGAATAGGCAATGAGACTTGGGGAGATCCCAGAGTCATAAAAGTCATCACAGAGCCGATCCCAGTTTCTGATCTCCGTGTTGCCCTCACGGGTGTGAGGAAGGCTGCTCTCTCCTGGAGCAATGGCAATGGCACTGCCTCCTGCCGGGTTCTTCTTGAAAGCATTGGAAGCCATGAGGAGTTGACTCAAGACTCAAGACTTCAGGTCAATATCTCGGGCCTGAAGCCAGGGGTTCAATACAACATCAACCCGTATCTTCTACAATCAAATAAGACAAAGGGAGACCCCTTGGGCACAGAAGGTGGCTTGGATGCCAGCAATACAGAGAGAAGCCGGGCAGGGAGCCCCACCGCCCCTGTGCATGATGAGTCCCTCGTGGGACCTGTGGACCCATCCTCCGGCCAGCAGTCCCGAGACACGGAAGTCCTGCTTGTCGGGTTAGAGCCTGGCACCCGATACAATGCCACCGTTTATTCCCAAGCAGCGAATGGCACAGAAGGACAGCCCCAGGCCATAGAGTTCAGGACAAATGCTATTCAGGTTTTTGACGTCACCGCTGTGAACATCAGTGCCACAAGCCTGACCCTGATCTGGAAAGTCAGCGATAACGAGTCGTCATCTAACTATACCTACAAGATACATGTGGCGGGGGAGACAGATTCTTCCAATCTCAACGTCAGTGAGCCTCGCGCTGTCATCCCCGGACTCCGCTCCAGCACCTTCTACAACATCACAGTGTGTCCTGTCCTAGGTGACATCGAGGGCACGCCGGGCTTCCTCCAAGTGCACACCCCCCCTGTTCCAGTTTCTGACTTCCGAGTGACAGTGGTCAGCACGACGGAGATCGGCTTAGCATGGAGCAGCCATGATGCAGAATCATTTCAGATGCATATCACACAGGAGGGAGCTGGCAATTCTCGGGTAGAAATAACCACCAACCAAAGTATTATCATTGGTGGCTTGTTCCCTGGAACCAAGTATTGCTTTGAAATAGTTCCAAAAGGACCAAATGGGACTGAAGGGGCATCTCGGACAGTTTGCAATAGAACTGTTCCCAGTGCAGTGTTTGACATCCACGTGGTCTACGTCACCACCACGGAGATGTGGCTGGACTGGAAGAGCCCTGACGGTGCTTCCGAGTATGTCTACCATTTAGTCATAGAGTCCAAGCATGGCTCTAACCACACAAGCACGTATGACAAAGCGATTACTCTCCAGGGCCTGATTCCGGGCACCTTATATAACATCACCATCTCTCCAGAAGTGGACCACGTCTGGGGGGACCCCAACTCCACTGCACAGTACACACGGCCCAGCAATGTGTCCAACATTGATGTAAGTACCAACACCACAGCAGCAACTTTAAGTTGGCAGAACTTTGATGACGCCTCTCCCACGTACTCCTACTGCCTTCTTATTGAGAAGGCTGGAAATTCCAGCAACGCAACACAAGTAGTCACGGACATTGGAATTACTGACGCTACAGTCACTGAATTAATACCTGGCTCATCATACACAGTGGAGATCTTTGCACAAGTAGGGGATGGGATCAAGTCACTGGAACCTGGCCGGAAGTCATTCTGTACAGATCCTGCGTCCATGGCCTCCTTCGACTGCGAAGTGGTCCCCAAAGAGCCAGCCCTGGTTCTCAAATGGACCTGCCCTCCTGGCGCCAATGCAGGCTTTGAGCTGGAGGTCAGCAGTGGAGCCTGGAACAATGCGACCCACCTGGAGAGCTGCTCCTCTGAGAATGGCACTGAGTATAGAACGGAAGTCACGTATTTGAATTTTTCTACCTCGTACAACATCAGCATCACCACTGTGTCCTGTGGAAAGATGGCAGCCCCCACCCGGAACACCTGCACTACTGGCATCACAGATCCCCCTCCTCCAGATGGATCCCCTAATATTACATCTGTCAGTCACAATTCAGTAAAGGTCAAGTTCAGTGGATTTGAAGCCAGCCACGGACCCATCAAAGCCTATGCTGTCATTCTCACCACCGGGGAAGCTGGTCACCCTTCTGCAGATGTCCTGAAATACACGTATGAGGATTTCAAAAAGGGAGCCTCAGATACTTATGTGACATACCTCATAAGAACAGAAGAAAAGGGACGTTCTCAGAGCTTGTCTGAAGTTTTGAAATATGAAATTGACGTTGGGAATGAGTCAACCACACTTGGTTATTACAATGGGAAGCTGGAACCTCTGGGCTCCTACCGGGCTTGTGTGGCTGGCTTCACCAACATTACCTTCCACCCTCAAAACAAGGGGCTCATTGATGGGGCTGAGAGCTATGTGTCCTTCAGTCGCTACTCAGATGCTGTTTCCTTGCCCCAGGATCCAGGTGTCATCTGTGGCGGTGGACAC CACCATCATCACCATCACCACCATCACTAG

HPTP-β, cyno PTP-β, and HPTP-η proteins were coated onto ELISA plates incoating buffer (pH 9.4). Each protein was prepared as a 1 μg/mL proteinstock in 50 mM carbonate-bicarbonate. The coated proteins were aliquotedat a volume of 100 μL/well into sterile, clear, polystyrene, flat bottom96-well plates. The plates were incubated overnight at 4° C. Afterovernight incubation, the plates were washed 3× with 200 μL/well of 1×PBS-T (PBS with 0.05% Tween 20). The immunizing protein was human VE-PTP(HPTP-β). The plates were then blocked with 200 μL/well of 5% blottinggrade blocker non-fat dry milk in PBS-T and incubated for 1 hr at roomtemperature.

Primary antibody samples were prepared for the humanized variants andthe negative control, anti-hCD20-hIgG4S228P, using 1 μg/well in 5%blotting grade blocker non-fat dry milk in PBS. Samples were tested intriplicates. The Fab antibody fragments were treated as the IgGantibodies. The plates were then washed 3× with PBS-T and 100 μL ofprimary antibody preparation was added in each well. The plates werethen incubated for 1 hr at room temperature.

Secondary antibody samples were prepared by dilutingperoxidase-conjugated affinity purified donkey anti-human IgG specificsecondary antibody at 1:2,000 dilution in blotting grade blocker non-fatdry milk in PBS. The plates were then washed 3× with PBS-T and 100 μL ofthe secondary antibody preparation was added in each well. The plateswere then incubated for 1 hr at room temperature. After incubation, theplates were washed 3× with PBS-T.

Then, 75 μg/1-Step™ Ultra TMB-ELISA was added in each well, and theplate was allowed to develop for 5 min at room temperature. After 5 min,50 μL of 2 M sulfuric acid was added to each well to stop the reaction.The plates were then analyzed on a plate reader at an absorbancewavelength of 450 nm.

FIG. 5 illustrates western blots of R15E6 screened againstspecies-specific VE-PTP and HPTP-η. R15E6 was highly selective for humanVE-PTP (HPTP-β; lanes 1 and 4). No significant binding to human PTP-η orcyno VE-PTP (cyno PTP-β) was observed. The indicated recombinanthexahistidine-tagged proteins (6-His) (SEQ ID NO: 52) were probedsequentially with R15E6 and a commercially-available 6-His antibody(“6-His” is SEQ ID NO: 52). The commercially-available 6-His antibody(“6-His” is SEQ ID NO: 52) exhibited binding to all protein targets, andshowed poor selectivity for human VE-PTP (HPTP-β).

Each humanized variant was measured for affinity to the antigen, humanVE-PTP (HPTP-β), using ELISA. 100 mg/well of the VE-PTP (HPTP-β) antigenwere immobilized onto 96-well Maxisorp plates in coating buffer (0.5 mMNaHCO₃ brought to pH 9.6 by the addition of 0.5 mM Na₂CO₃) overnight at4° C. The coating buffer was then removed and 200 μL/well of blocksolution (3% w/v semi-skimmed milk powder, PBS) was added and agitatedfor 2 hrs at room temperature. The plate was washed four times withPBS-T (1% v/v Tween 20). 100 μL/well was added of purified R15E6variants serially diluted from 10,000 ng/mL to 9.765 ng/mL in PBS-TB(0.05% v/v Tween 20, 0.5% w/v BSA). Following agitation for 2 hrs atroom temperature, the plate was washed 4× with PBS-T. 100 μL/well goatanti-human HRP (Fc specific) at a ratio of 1:60,000 PBS was then addedand the plates were incubated for an additional hour with agitation atroom temperature. The plate was then washed 6× with PBS-T and once inPBS. Next, 100 μL/well of TMB substrate solution was added and incubatedat 37° C. for 10 min. 50 μL 1 M HCl was added per well and the plateimmediately read at 450 nm on a Tecan Sunrise plate reader. The ELISAresults are shown in FIGS. 14-17. The HC1, HC2 and HC3 variantsgenerally displayed similar binding characteristics compared to thechimeric HC0LC0 antibody. The binding of the HC4 variants was much lowerthan with the other heavy chains variants compared to HC0LC0 (FIG. 17).

Samples of each variant were further assessed by Biacore analysis todetermine the binding affinity. Antibody binding experiments wereperformed on Biacore 3000™ at 25° C. Assay buffer: 10 mM HEPES buffer(pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% P20 (polyoxyethylenesorbitan).Regeneration buffer: 10 mM glycine HCl (pH 1.75). Conjugation buffer: 10mM sodium acetate buffer (pH 5). The flow rate used for capturing theligand was 5 μL/min. The flow rate for the kinetic analysis was 50μL/min. Flow cell 1 and 2 of the CMS chip were coated with maximumamounts of goat anti-human IgG. Approximately 150 RU of the testantibody was captured on flow cell 2, 3, and 4 as indicated. The antigenwas flowed over the chip. Binding of the antigen to the antibodies wasmonitored in real time. The K_(D) was determined from the observedk_(on) and k_(off) values.

A scouting analysis was performed using single analyte concentrations asindicated. At these concentrations, binding can be observed even if theligand binding is weak. Flow cell 1 response was used for referencesubtraction.

A full kinetic analysis was performed for the control antibody with a2-fold serial dilution with the range of concentrations the analyte asindicated: 50, 25, 12.5, 6.25, 3.125, and 0 nM.

TABLE 17 shows the binding kinetic parameters of the binding of eachhumanized antibody to VE-PTP (HPTP-β) ECD, including the associationrate (k_(a)), dissociation rate (k_(d)), maximum binding capacity(R_(max)), the association constant (K_(A)), and the dissociationconstant (K_(D)) as determined by Biacore binding assays. R_(max) wasmeasured as the relative response (RU). All of the humanized candidatesexhibited similar binding affinities as the mouse/human chimericantibody, HC0LC0.

TABLE 17 R_(max) Conc. Of Ligand Analyte k_(a) (1/MS) k_(d) (1/s) (RU)Analyte K_(A) (1/M) K_(D) (M) HC0:LC0 ½ VE-PTP 2.09 × 10⁵ 6.56 × 10⁻⁴27.2 50-0 nM 3.18 × 10⁸  3.14 × 10⁻⁹  (150RU) ECD 6.71 × 10⁴ 4.17 × 10⁻⁴69.6  100 nM 1.61 × 10⁸  6.22 × 10⁻⁹  HC2:LC4 ½ VE-PTP 9.86 × 10⁵ 4.29 ×10⁻⁴ 13.9 50-0 nM 2.30 × 10⁹  4.36 × 10⁻¹⁰ (145RU) ECD 5.15 × 10⁴ 6.68 ×10⁻⁴ 67.3  100 nM 7.72 × 10⁷  1.30 × 10⁻⁸  HC2:LC1 ½ VE-PTP 1.08 × 10⁶7.23 × 10⁻⁵ 8.38 50-0 nM  1.5 × 10¹⁰ 6.68 × 10⁻¹¹ (140RU) ECD 4.98 × 10⁴6.88 × 10⁻⁴ 54.2  100 nM 7.24 × 10⁷  1.38 × 10⁻⁸  HC1:LC2 ½ VE-PTP 4.10× 10⁵ 9.47 × 10⁻⁴ 29.6 50-0 nM 4.33 × 10⁸  2.31 × 10⁻⁹  (150RU) ECD 2.51× 10⁵ 7.69 × 10⁻⁴ 52.4  100 nM 3.27 × 10⁸  3.06 × 10⁻⁹  HC2:LC3 ½ VE-PTP8.90 × 10⁵ 1.23 × 10⁻³ 22.6 25-0 nM 6.75 × 10⁸  1.48 × 10⁻⁹  (145RU) ECD2.22 × 10⁵ 6.62 × 10⁻⁴ 38.8  100 nM 3.36 × 10⁸  2.98 × 10⁻⁹  HC3:LC2 ½VE-PTP 5.39 × 10⁴ 1.23 × 10⁻³ 104 25-0 nM 4.30 × 10⁷  2.32 × 10⁻⁸ (140RU) ECD 2.05 × 10⁵ 9.93 × 10⁻⁴ 36.8  100 nM 2.06 × 10⁸  4.84 × 10⁻⁹ HC1:LC4 ½ VE-PTP 5.96 × 10⁵ 1.34 × 10⁻³ 24.6 50-0 nM 4.44 × 10⁸  2.25 ×10⁻⁹  (150RU) ECD 1.79 × 10⁵ 7.92 × 10⁻⁴ 49  100 nM 2.26 × 10⁸  4.42 ×10⁻⁹  HC2:LC2 ½ VE-PTP 4.25 × 10⁴ 2.15 × 10⁻³ 313 25-0 nM 1.98 × 10⁷ 5.06 × 10⁻⁸  (145RU) ECD 1.49 × 10⁵ 5.24 × 10⁻⁴ 40.8  100 nM 2.85 × 10⁸ 3.51 × 10⁻⁹  HC3:LC4 ½ VE-PTP 1.34 × 10⁵ 1.09 × 10⁻³ 42.8 25-0 nM 1.23 ×10⁸  8.14 × 10⁻⁹  (140RU) ECD 1.39 × 10⁵ 8.87 × 10⁻⁴ 44.7  100 nM 1.59 ×10⁸  6.29 × 10⁻⁹ 

TABLE 18 shows the binding selectivity of the humanized antibodiescompared to the parental antibody (R15E6) and the inactive control(IgG4) as determined by ELISA. A value that is less than or equal to 0.2signifies no binding. The humanized antibody candidates, the parentalR15E6, and the mouse/human chimeric antibody (HC0:LC0) all exhibitedhigh selectivity for human VE-PTP (HPTP-β) as indicated by nosignificant binding to human PTP-η and cyno PTP-β.

TABLE 18 Clone Human Human Cyno (1 μg/well) VE-PTP PTPη VE-PTP HC0:LC01.8 0.16 0.12 HC1:LC2 1.75 0.12 0.13 HC1:LC4 1.74 0.12 0.19 HC2:LC1 1.810.13 0.18 HC2:LC2 1.92 0.12 0.12 HC2:LC3 1.55 0.12 0.13 HC2:LC4 1.650.11 0.13 HC3:LC4 2.00 0.11 0.14 Parental 2.43 0.14 0.17 IgG4 0.20 0.130.16

The humanized antibodies used for targeting human VE-PTP (HPTP-β) canrestore Tie2 activity by autophosphorylation and initiate downstreameffectors including, for example, proteins in the PI3K-Akt pathway. Inaddition to assessing binding of the antibodies to human VE-PTP(HPTP-β), the ability of the antibodies to activate these effectorproteins was measured. FIG. 18 illustrates the activation of Tie2 (leftpanel) and Akt (right panel) in the absence of Tie2 ligands (e.g., Ang1or Ang2) by the humanized variants as determined by western blot. Theactivities of the humanized variants were similar to that of theparental murine antibody R15E6 and the chimeric antibody HC0LC0. HumanIgG4 was used as a control. All antibodies were tested at 50 nM. The toppanel of each blot shows phosphorylation of either Tie2 or Akt,indicating Tie2 or Akt activation. Tie2 and Akt exhibited greaterphosphorylation in samples treated with the humanized antibodies thanthose samples treated with the control antibodies, and indicated thatthe humanized antibodies were effective in activating Tie2 and Akt evenin the absence of ligands.

FIG. 19 illustrates concentration-dependent activation of Tie2 (toppanel) and Akt (bottom panel) in the absence of Tie2 ligands (e.g., Ang1or Ang2) by humanized antibodies, HC2LC4 and HC1LC1 as determined bywestern blot. Tie2 and Akt signaling activity was observed at lownanomolar concentrations of the humanized variants. The upper panel ofeach blot shows phosphorylation of either Tie2 or Akt, indicating Tie2or Akt activation. Tie2 and Akt exhibited greater phosphorylation in thesamples treated with the humanized antibodies than exhibited in samplestreated with the control antibodies. This result indicates that thehumanized antibodies were effective in activating Tie2 and Akt even inthe absence of Ang1.

FIG. 20 illustrates the activation of Tie2 (left panel) and Akt (rightpanel) in the presence of Ang1 and Ang2 by the humanized antibodycandidates, HC2LC4 and HC1LC1, as determined by western blot. HC2LC4 andHC1LC1 activated both Tie2 and Akt in the presence of Ang2, and enhancedAng1-mediated Tie2 and Akt activation, as indicated by the increase inTie2 and Akt phosphorylation, respectively. All antibodies were testedat 10 nM.

FIG. 21 illustrates the activation of Tie2 (panel A), human VE-PTP(HPTP-β) expression (panel B), and Akt (panel C) in the presence of Ang1or Ang2 by the humanized antibody candidates, HC2LC1 and HC2LC4, asdetermined by western blot. HC2LC1 and HC2LC4 activated Tie2 in thepresence of Ang1 alone, Ang2 alone, and in the presence of both Ang1 andAng2. The antibodies were pre-incubated for 15 min and then stimulatedby Ang1 or Ang2 for 10 min at a concentration of 600 ng/mL. Allantibodies were tested at 10 nM.

FIG. 22 illustrates the activation of Akt in HUVECs using the humanizedantibody candidate, HC2LC1. The upper panel of the first 3 blots showsphosphorylation of Akt, indicating Akt activation, at increasingconcentrations of HC2LC1. Consistent with the binding affinity study,HC2LC1 activated Akt at nanomolar concentrations of antibody. Aktactivation by HC2LC1 was blocked by pre-incubation of the antibody withthe human VE-PTP (HPTP-β) extracellular domain (βECD 6His (“6-His” isSEQ ID NO: 52) and βECD Fc). This result indicates that HC2LC1-mediatedAkt activation occurs through HC2LC1 binding to the human VE-PTP(HPTP-β) extracellular domain.

FIG. 23 illustrates the enhanced viability in serum-starved HUVECs usinghumanized antibody candidates HC2LC1 and HC0LC0, as compared to theparental antibody, R15E6, and the mIgG1 control.

EMBODIMENTS

Embodiment 1. A compound comprising a sequence that is at least 80%identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:12.

Embodiment 2. The compound of embodiment 1, wherein the sequence is atleast 85% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, orSEQ ID NO: 12.

Embodiment 3. The compound of any one of embodiments 1-2, wherein thesequence is at least 90% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 11, or SEQ ID NO: 12.

Embodiment 4. The compound of any one of embodiments 1-3, wherein thesequence is at least 95% identical to SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 11, or SEQ ID NO: 12.

Embodiment 5. The compound of embodiment 1, wherein the sequence is atleast 80% identical to SEQ ID NO: 9.

Embodiment 6. The compound of any one of embodiments 1-5, wherein thesequence is SEQ ID NO: 9.

Embodiment 7. The compound of embodiment 1, wherein the sequence is atleast 80% identical to SEQ ID NO: 10.

Embodiment 8. The compound of any one of embodiments 1-4 and 7, whereinthe sequence is SEQ ID NO: 10.

Embodiment 9. The compound of embodiment 1, wherein the sequence is atleast 80% identical to SEQ ID NO: 11.

Embodiment 1. The compound of any one of embodiments 1-4 and 9, whereinthe sequence is SEQ ID NO: 11.

Embodiment 11. The compound of embodiment 1, wherein the sequence is atleast 80% identical to SEQ ID NO: 12.

Embodiment 12. The compound of any one of embodiments 1-4 and 11,wherein the sequence is SEQ ID NO: 12.

Embodiment 13. The compound of embodiment 1, wherein the sequence is atleast 80% identical to SEQ ID NO: 29.

Embodiment 14. The compound of embodiment 1, wherein the sequence is SEQID NO: 29.

Embodiment 15. The compound of embodiment 1, wherein the sequence is atleast 80% identical to SEQ ID NO: 30.

Embodiment 16. The compound of embodiment 1, wherein the sequence is SEQID NO: 30.

Embodiment 17. The compound of embodiment 1, wherein the sequence is atleast 80% identical to SEQ ID NO: 31.

Embodiment 18. The compound of embodiment 1, wherein the sequence is SEQID NO:

Embodiment 19. The compound of embodiment 1, wherein the sequence is atleast 80% identical to SEQ ID NO: 32.

Embodiment 20. The compound of embodiment 1, wherein the sequence is SEQID NO: 32.

Embodiment 21. The compound of any one of embodiments 1-20, wherein thecompound inhibits a tyrosine phosphatase.

Embodiment 22. The compound of any one of embodiments 1-21, wherein thecompound inhibits HPTP-β.

Embodiment 23. The compound of any one of embodiments 1-22, wherein thecompound inhibits VE-PTP.

Embodiment 24. The compound of any one of embodiments 1-23, wherein thecompound activates Tie2.

Embodiment 25. The compound of any one of embodiments 1-24, wherein thecompound activates Akt.

Embodiment 26. The compound of any one of embodiments 1-25, wherein thecompound binds an extracellular domain of HPTP-β.

Embodiment 27. The compound of any one of embodiments 1-26, wherein thecompound binds the first FN3 repeat of an extracellular domain ofHPTP-β.

Embodiment 28. The compound of any one of embodiments 1-27, wherein abinding affinity (K_(D)) of the compound to the extracellular domain ofHPTP-β is about 70 pM to about 70 nM.

Embodiment 29. A compound comprising a sequence that is at least 80%identical to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO:23.

Embodiment 30. The compound of embodiment 29, wherein the sequence is atleast 85% identical to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, orSEQ ID NO: 23.

Embodiment 31. The compound of any one of embodiments 29-30, wherein thesequence is at least 90% identical to SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, or SEQ ID NO: 23.

Embodiment 32. The compound of any one of embodiments 29-31, wherein thesequence is at least 95% identical to SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, or SEQ ID NO: 23.

Embodiment 33. The compound of embodiment 29, wherein the sequence is atleast 80% identical to SEQ ID NO: 20.

Embodiment 34. The compound of any one of embodiments 29-33, wherein thesequence is SEQ ID NO: 20.

Embodiment 35. The compound of embodiment 29, wherein the sequence is atleast 80% identical to SEQ ID NO: 21.

Embodiment 36. The compound of any one of embodiments 29-32 and 35,wherein the sequence is SEQ ID NO: 21.

Embodiment 37. The compound of embodiment 29, wherein the sequence is atleast 80% identical to SEQ ID NO: 22.

Embodiment 38. The compound of any one of embodiments 29-32 and 37,wherein the sequence is SEQ ID NO: 22.

Embodiment 39. The compound of embodiment 29, wherein the sequence is atleast 80% identical to SEQ ID NO: 23.

Embodiment 40. The compound of any one of embodiments 29-32 and 39,wherein the sequence is SEQ ID NO: 23.

Embodiment 41. The compound of embodiment 29, wherein the sequence is atleast 80% identical to SEQ ID NO: 34.

Embodiment 42. The compound of embodiment 29, wherein the sequence isSEQ ID NO: 34.

Embodiment 43. The compound of embodiment 29, wherein the sequence is atleast 80% identical to SEQ ID NO: 35.

Embodiment 44. The compound of embodiment 29, wherein the sequence isSEQ ID NO: 35.

Embodiment 45. The compound of embodiment 29, wherein the sequence is atleast 80% identical to SEQ ID NO: 36.

Embodiment 46. The compound of embodiment 29, wherein the sequence isSEQ ID NO: 36.

Embodiment 47. The compound of embodiment 29, wherein the sequence is atleast 80% identical to SEQ ID NO: 37.

Embodiment 48. The compound of embodiment 29, wherein the sequence isSEQ ID NO: 37.

Embodiment 49. The compound of any one of embodiments 29-48, wherein thecompound inhibits a tyrosine phosphatase.

Embodiment 50. The compound of any one of embodiments 29-49, wherein thecompound inhibits HPTP-β.

Embodiment 51. The compound of any one of embodiments 29-50, wherein thecompound inhibits VE-PTP.

Embodiment 52. The compound of any one of embodiments 29-51, wherein thecompound activates Tie2.

Embodiment 53. The compound of any one of embodiments 29-52, wherein thecompound activates Akt.

Embodiment 54. The compound of any one of embodiments 29-53, wherein thecompound binds an extracellular domain of HPTP-β.

Embodiment 55. The compound of any one of embodiments 29-54, wherein thecompound binds the first FN3 repeat of an extracellular domain ofHPTP-β.

Embodiment 56. The compound of any one of embodiments 29-55, wherein abinding affinity (K_(D)) of the compound to the extracellular domain ofHPTP-β is about 70 pM to about 70 nM.

Embodiment 57. A compound comprising: a) a heavy chain that comprises asequence that is at least 80% identical to SEQ ID NO: 30; and b) a lightchain that comprises a sequence that is at least 80% identical to SEQ IDNO: 34.

Embodiment 58. The compound of embodiment 57, wherein the heavy chain isat least 85% identical to SEQ ID NO: 30; and the light chain is at least85% identical to SEQ ID NO: 34.

Embodiment 59. The compound of any one of embodiments 57-58, wherein theheavy chain is at least 90% identical to SEQ ID NO: 30; and the lightchain is at least 90% identical to SEQ ID NO: 34.

Embodiment 60. The compound of any one of embodiments 57-59, wherein theheavy chain is at least 95% identical to SEQ ID NO: 30; and the lightchain is at least 95% identical to SEQ ID NO: 34.

Embodiment 61. The compound of any one of embodiments 57-60, wherein theheavy chain is SEQ ID NO: 30 and the light chain is SEQ ID NO: 34.

Embodiment 62. The compound of any one of embodiments 57-61, wherein thecompound inhibits a tyrosine phosphatase.

Embodiment 63. The compound of any one of embodiments 57-62, wherein thecompound inhibits HPTP-β.

Embodiment 64. The compound of any one of embodiments 57-63, wherein thecompound inhibits VE-PTP.

Embodiment 65. The compound of any one of embodiments 57-64, wherein thecompound activates Tie2.

Embodiment 66. The compound of any one of embodiments 57-65, wherein thecompound activates Akt.

Embodiment 67. The compound of any one of embodiments 57-66, wherein thecompound binds an extracellular domain of HPTP-β.

Embodiment 68. The compound of any one of embodiments 57-67, wherein thecompound binds the first FN3 repeat of an extracellular domain ofHPTP-β.

Embodiment 69. The compound of any one of embodiments 57-68, wherein abinding affinity (K_(D)) of the compound to the extracellular domain ofHPTP-β is about 70 pM to about 70 nM.

Embodiment 70. A compound comprising: a) a heavy chain that comprises asequence that is at least 80% identical to SEQ ID NO: 29; and b) a lightchain that comprises a sequence that is at least 80% identical to SEQ IDNO: 35.

Embodiment 71. The compound of embodiment 70, wherein the heavy chain isat least 85% identical to SEQ ID NO: 29; and the light chain is at least85% identical to SEQ ID NO: 35.

Embodiment 72. The compound of any one of embodiments 70-71, wherein theheavy chain is at least 90% identical to SEQ ID NO: 29; and the lightchain is at least 90% identical to SEQ ID NO: 35.

Embodiment 73. The compound of any one of embodiments 70-72, wherein theheavy chain is at least 95% identical to SEQ ID NO: 29; and the lightchain is at least 95% identical to SEQ ID NO: 35.

Embodiment 74. The compound of any one of embodiments 70-73, wherein theheavy chain is SEQ ID NO: 29 and the light chain is SEQ ID NO: 35.

Embodiment 75. The compound of any one of embodiments 70-74, wherein thecompound inhibits a tyrosine phosphatase.

Embodiment 76. The compound of any one of embodiments 70-75, wherein thecompound inhibits HPTP-β.

Embodiment 77. The compound of any one of embodiments 70-76, wherein thecompound inhibits VE-PTP.

Embodiment 78. The compound of any one of embodiments 70-77, wherein thecompound activates Tie2.

Embodiment 79. The compound of any one of embodiments 70-78, wherein thecompound activates Akt.

Embodiment 80. The compound of any one of embodiments 70-79, wherein thecompound binds an extracellular domain of HPTP-β.

Embodiment 81. The compound of any one of embodiments 70-80, wherein thecompound binds the first FN3 repeat of an extracellular domain ofHPTP-β.

Embodiment 82. The compound of any one of embodiments 70-81, wherein abinding affinity (K_(D)) of the compound to the extracellular domain ofHPTP-β is about 70 pM to about 70 nM.

Embodiment 83. A compound comprising: a) a heavy chain that comprises asequence that is at least 80% identical to SEQ ID NO: 30; and b) a lightchain that comprises a sequence that is at least 80% identical to SEQ IDNO: 37.

Embodiment 84. The compound of embodiment 83, wherein the heavy chain isat least 85% identical to SEQ ID NO: 30; and the light chain is at least85% identical to SEQ ID NO: 37.

Embodiment 85. The compound of any one of embodiments 83-84, wherein theheavy chain is at least 90% identical to SEQ ID NO: 30; and the lightchain is at least 90% identical to SEQ ID NO: 37.

Embodiment 86. The compound of any one of embodiments 83-85, wherein theheavy chain is at least 95% identical to SEQ ID NO: 30; and the lightchain is at least 95% identical to SEQ ID NO: 37.

Embodiment 87. The compound of any one of embodiments 83-86, wherein theheavy chain is SEQ ID NO: 30 and the light chain is SEQ ID NO: 37.

Embodiment 88. The compound of any one of embodiments 83-87, wherein thecompound inhibits a tyrosine phosphatase.

Embodiment 89. The compound of any one of embodiments 83-88, wherein thecompound inhibits HPTP-β.

Embodiment 90. The compound of any one of embodiments 83-89, wherein thecompound inhibits VE-PTP.

Embodiment 91. The compound of any one of embodiments 83-90, wherein thecompound activates Tie2.

Embodiment 92. The compound of any one of embodiments 83-91, wherein thecompound activates Akt.

Embodiment 93. The compound of any one of embodiments 83-92, wherein thecompound binds an extracellular domain of HPTP-β.

Embodiment 94. The compound of any one of embodiments 83-93, wherein thecompound binds the first FN3 repeat of an extracellular domain ofHPTP-β.

Embodiment 95. The compound of any one of embodiments 83-94, wherein abinding affinity (K_(D)) of the compound to the extracellular domain ofHPTP-β is about 70 pM to about 70 nM.

Embodiment 96. A method of treating a condition in a subject in needthereof, the method comprising administering to the subject atherapeutically-effective amount of the compound of any one ofembodiments 1-95.

Embodiment 97. The method of embodiment 96, wherein the condition is anocular condition.

Embodiment 98. The method of embodiment 96 or 97, wherein the conditionis diabetic retinopathy.

Embodiment 99. The method of any one of embodiments 96-97, wherein thecondition is neovascularization.

Embodiment 100. The method of any one of embodiments 96-97, wherein thecondition is vascular leak.

Embodiment 101. The method of any one of embodiments 96-97, wherein thecondition is increased intraocular pressure.

Embodiment 102. The method of any one of embodiments 96-97, wherein thecondition is ocular edema.

Embodiment 103. The method of embodiment 96 or 97, wherein the conditionis diabetic macular edema.

Embodiment 104. The method of any one of embodiment 96 or 97, whereinthe condition is ocular hypertension.

Embodiment 105. The method of embodiment 96 or 97, wherein the conditionis ocular inflammation.

Embodiment 106. The method of any one of embodiments 96-105, wherein theadministration is to an eye of the subject.

Embodiment 107. The method of any one of embodiments 96-106, wherein theadministration is intravitreal.

Embodiment 108. The method of any one of embodiments 96-105, wherein theadministration is subcutaneous.

Embodiment 109. The method of any one of embodiments 96-106, wherein theadministration is topical.

Embodiment 110. The method of any one of embodiments 96-109, wherein thesubject is a human.

Embodiment 111. The method of any one of embodiments 96-110, wherein thetherapeutically-effective amount of the compound is from about 0.25 mgto about 200 mg.

Embodiment 112. The method of any one of embodiments 96-110, wherein thetherapeutically-effective amount of the compound is from about 1 mg/kgto about 10 mg/kg.

Embodiment 113. The method of any one of embodiments 96-110, wherein thetherapeutically-effective amount of the compound is from about 1 mg toabout 50 mg.

Embodiment 114. The method of any one of embodiments 96-110, wherein thetherapeutically-effective amount of the compound is from about 50 mg toabout 200 mg.

What is claimed is:
 1. An antibody comprising: a heavy chain variable domain sequence that is any one of SEQ ID NOs: 9-12, wherein the heavy chain variable domain sequence comprises complementarity-determining region (CDR) sequences that are SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, wherein heavy chain CDR1 comprises SEQ ID NO: 54, heavy chain CDR2 comprises SEQ ID NO: 55, and heavy chain CDR3 comprises SEQ ID NO: 56, wherein the heavy chain variable domain sequence is contained within a heavy chain sequence that is any one of SEQ ID NOs: 29-32; and a light chain variable domain sequence that is any one of SEQ ID NOs: 20-23, wherein the light chain variable domain sequence comprises CDR sequences that are SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, wherein light chain CDR1 comprises SEQ ID NO: 57, light chain CDR2 comprises SEQ ID NO: 58, and light chain CDR3 comprises SEQ ID NO: 59, wherein the light chain variable domain sequence is contained within a light chain sequence that is any one of SEQ ID NOs: 34-37, wherein the antibody binds to human protein tyrosine phosphatase-beta (HPTP-β).
 2. The antibody of claim 1, wherein the antibody binds an extracellular domain of HPTPβ.
 3. The antibody of claim 1, wherein a binding affinity (K_(D)) of the antibody to the extracellular domain of HPTP-β is about 70 pM to about 70 nM.
 4. The antibody of claim 1, wherein the antibody binds a first FN3 repeat of an extracellular domain of HPTP-β.
 5. The antibody of claim 1, wherein the antibody inhibits HPTP-β.
 6. The antibody of claim 1, wherein the antibody activates tyrosine kinase with immunoglobulin and epidermal growth factor homology domains 2 (Tie-2).
 7. The antibody of claim 1, wherein the antibody activates Protein kinase B (Akt).
 8. The antibody of claim 1, wherein the antibody is humanized.
 9. The antibody of claim 1, wherein the heavy chain variable domain sequence is SEQ ID NO:
 9. 10. The antibody of claim 1, wherein the heavy chain variable domain sequence is SEQ ID NO:
 10. 11. The antibody of claim 1, wherein the heavy chain variable domain sequence is SEQ ID NO:
 11. 12. The antibody of claim 1, wherein the heavy chain variable domain sequence is SEQ ID NO:
 12. 13. The antibody of claim 1, wherein the light chain variable domain sequence is SEQ ID NO:
 20. 14. The antibody of claim 1, wherein the light chain variable domain sequence is SEQ ID NO:
 21. 15. The antibody of claim 1, wherein the light chain variable domain sequence is SEQ ID NO:
 22. 16. The antibody of claim 1, wherein the light chain variable domain sequence is SEQ ID NO:
 23. 17. A pharmaceutical composition comprising a therapeutically effective amount of an antibody comprising: a heavy chain variable domain sequence that is any one of SEQ ID NOs: 9-12, wherein the heavy chain variable domain sequence comprises complementarity-determining region (CDR) sequences that are SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, wherein heavy chain CDR1 comprises SEQ ID NO: 54, heavy chain CDR2 comprises SEQ ID NO: 55, and heavy chain CDR3 comprises SEQ ID NO: 56, wherein the heavy chain variable domain sequence is contained within a heavy chain sequence that is any one of SEQ ID NOs: 29-32; a light chain variable domain sequence that is any one of SEQ ID NOs: 20-23, wherein the light chain variable domain sequence comprises CDR sequences that are SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, wherein light chain CDR1 comprises SEQ ID NO: 57, light chain CDR2 comprises SEQ ID NO: 58, and light chain CDR3 comprises SEQ ID NO: 59, wherein the light chain variable domain sequence is contained within a light chain sequence that is any one of SEQ ID NOs: 34-37; and a pharmaceutically acceptable carrier.
 18. The composition of claim 17, wherein the therapeutically effective amount of the antibody is from about 0.25 mg to about 200 mg.
 19. The composition of claim 17, wherein the therapeutically effective amount of the antibody is from about 1 mg/kg to about 10 mg/kg.
 20. The composition of claim 17, wherein the therapeutically effective amount of the antibody is from about 1 mg to about 50 mg.
 21. The composition of claim 17, wherein the therapeutically effective amount of the antibody is from about 50 mg to about 200 mg.
 22. The composition of claim 17, wherein the composition is formulated for intravitreal administration.
 23. The composition of claim 17, wherein the composition is formulated for intravenous administration.
 24. The composition of claim 17, wherein the composition is formulated for subcutaneous administration.
 25. The composition of claim 17, wherein the composition is formulated for topical administration.
 26. The composition of claim 17, wherein the heavy chain variable domain sequence is SEQ ID NO:
 9. 27. The composition of claim 17, wherein the heavy chain variable domain sequence is SEQ ID NO:
 10. 28. The composition of claim 17, wherein the heavy chain variable domain sequence is SEQ ID NO:
 11. 29. The composition of claim 17, wherein the heavy chain variable domain sequence is SEQ ID NO:
 12. 30. The composition of claim 17, wherein the light chain variable domain sequence is SEQ ID NO:
 20. 31. The composition of claim 17, wherein the light chain variable domain sequence is SEQ ID NO:
 21. 32. The composition of claim 17, wherein the light chain variable domain sequence is SEQ ID NO:
 22. 33. The composition of claim 17, wherein the light chain variable domain sequence is SEQ ID NO:
 23. 